Thiol-based NAALADase inhibitors

ABSTRACT

This invention relates to new compounds, pharmaceutical compositions and diagnostic kits comprising such compounds, and methods of using such compounds for inhibiting NAALADase enzyme activity, detecting diseases where NAALADase levels are altered, effecting neuronal activity, effecting TGF-beta activity, inhibiting angiogenesis, and treating glutamate abnormalities, diabetic neuropathy, pain, compulsive disorders, prostate diseases, cancers and glaucoma.

This application is a divisional of U.S. application Ser. No.10/046,917, filed Jan. 17, 2002, now U.S. Pat. No. 6,586,623 which inturn claims the benefit of U.S. Provisional Application No. 60/261,754,filed Jan. 17, 2001, and U.S. Provisional Application No. 60/342,772,filed Dec. 28, 2001, the entire contents of which applications areherein incorporated by reference.

This invention relates to new compounds, pharmaceutical compositions anddiagnostic kits comprising such compounds, and methods of using suchcompounds for inhibiting NAALADase enzyme activity, detecting diseaseswhere NAALADase levels are altered, effecting neuronal activity,effecting TGF-β activity, inhibiting angiogenesis, and treatingglutamate abnormalities, diabetic neuropathy, pain, compulsivedisorders, prostate diseases, cancers and glaucoma.

The NAALADase enzyme, also known as prostate specific membrane antigen(“PSM” or “PSMA”) and human glutamate carboxypeptidase II (“GCP II”),catalyzes the hydrolysis of the neuropeptide N-acetyl-aspartyl-glutamate(“NAAG”) to N-acetyl-aspartate (“NAA”) and glutamate. Based upon aminoacid sequence homology, NAALADase has been assigned to the M28 family ofpeptidases.

Studies suggest NAALADase inhibitors may be effective in treatingischemia, spinal cord injury, demyelinating diseases, Parkinson'sdisease, Amyotrophic Lateral Sclerosis (“ALS”), alcohol dependence,nicotine dependence, cocaine dependence, cancer, diabetic neuropathy,pain and schizophrenia, and in inhibiting angiogenesis. In view of theirbroad range of potential applications, a need exists for new NAALADaseinhibitors and pharmaceutical compositions comprising such compounds.

SUMMARY OF THE INVENTION

This invention relates to a compound of formula I

or a pharmaceutically acceptable equivalent of said compound, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen or C₁-C₃alkyl;

A¹, A², A³ and A⁴ are independently hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy,halo, nitro, phenyl, phenoxy, benzyl, benzyloxy or —COOH, or anyadjacent two of A², A³ and A⁴ form with the benzene ring a fused 5- or6-membered carbocyclic or heterocyclic aromatic ring, said heterocyclicaromatic ring containing 1 or 2 oxygen, nitrogen and/or sulfurheteroatom(s).

This invention further relates to a compound of formula II

or a pharmaceutically acceptable equivalent of said compound, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen or C₁-C₃alkyl; and

A¹, A², A³, A⁴ and A⁵ are independently hydrogen, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₃ perhaloalkyl, phenyl, phenoxy, benzyl, benzyloxy, hydroxy,halo, cyano, nitro, —SO₂R⁹, —(C═O)NR⁹R¹⁰, —(C═O)NR⁹(CH₂)_(n)COOH,—NR⁹(C═O)R¹⁰, —(CH₂)_(n)COOH or —COOH, or any adjacent two of A¹, A²,A³, A⁴ and A⁵ form with the benzene ring a fused 5- or 6-memberedcarbocyclic or heterocyclic aromatic ring, said heterocyclic aromaticring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s);

R⁹ and R¹⁰ are independently hydrogen, C₁-C₆ alkyl, phenyl or benzyl;and

n is 1-3;

provided that if A¹, A³ and A⁵ are independently hydrogen, C₁-C₆ alkyl,C₁-C₆ alkoxy, halo, nitro, phenyl, phenoxy, benzyl, benzyloxy or —COOH,then neither A² nor A⁴ are —COOH; and provided that if any adjacent twoof A³, A⁴ and A⁵ form with the benzene ring a fused 5- or 6-memberedcarbocyclic or heterocyclic aromatic ring, said heterocyclic aromaticring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s),then A² is not —COOH.

This invention also relates to a compound of formula III

or a pharmaceutically acceptable equivalent of said compound, wherein:

X and Y are independently —CR⁵R⁶—, —O—, —S— or —NR—, provided that atleast one of X and Y is/are —CR⁵R⁶—;

A¹, A², A³, A⁴ and A⁵ are independently hydrogen, C₁-C₉ alkyl, C₂-C₉alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle, heterocycle, C₁-C₉alkoxy, C₂-C₉ alkenyloxy, phenoxy, benzyloxy, hydroxy, halo, nitro,cyano, isocyano, —COOR⁷, —COR⁷, —NR⁷R⁸, —SR⁷, —SOR⁷, —SO₂R⁷, —SO₂(OR⁷),—(C═O)NR⁷R⁸, —(C═O)NR⁷(CH₂)_(n)COOH, —NR⁷(C═O) R⁸ or —(CH₂)_(n)COOH, orany adjacent two of A¹, A², A³, A⁴ and A⁵ form with the benzene ring afused ring that is saturated or unsaturated, aromatic or non-aromatic,and carbocyclic or heterocyclic, said heterocyclic ring containing 1 or2 oxygen, nitrogen and/or sulfur heteroatom(s);

n is 1-3;

R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, C₁-C₉alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle orheterocycle; and

said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle,alkoxy, alkenyloxy, phenoxy, benzyloxy, and fused ring are independentlyunsubstituted or substituted with one or more substituent(s);

provided that if A¹, A² and A³ are each hydrogen, and A⁴ and A⁵ are each—COOH, then A⁴ is ortho to A⁵; and provided that if Y is —CR⁵R⁶—, thenat least one of A¹, A², A³, A⁴ and A⁵ is/are independently phenoxy,benzyloxy, aryl, heteroaryl, carbocycle or heterocycle that issubstituted with one or more substituent(s).

Additionally, this invention relates to method for inhibiting NAALADaseenzyme activity, treating a glutamate abnormality, effecting a neuronalactivity, treating a prostate disease, treating cancer, inhibitingangiogenesis or effecting a TGF-β activity, comprising administering toa mammal in need of such inhibition, treatment or effect, an effectiveamount of a compound of formula I, II or III, as described above.

This invention further relates to method for detecting a disease,disorder or condition where NAALADase levels are altered, comprising:

(i) contacting a sample of bodily tissue or fluid with a compound offormula I, II or III, as defined above, wherein said compound binds toany NAALADase in said sample; and

(ii) measuring the amount of any NAALADase bound to said sample, whereinthe amount of NAALADase is diagnostic for said disease, disorder orcondition.

This invention also relates to a method for detecting a disease,disorder or condition where NAALADase levels are altered in an animal ora mammal, comprising:

(i) labeling a compound of formula I, II or III, as defined above, withan imaging reagent;

(ii) administering to said animal or mammal an effective amount of thelabeled compound;

(iii) allowing said labeled compound to localize and bind to NAALADasepresent in said animal or mammal; and

(iv) measuring the amount of NAALADase bound to said labeled compound,wherein the amount of NAALADase is diagnostic for said disease, disorderor condition.

Additionally, this invention further relates to a diagnostic kit fordetecting a disease, disorder or condition where NAALADase levels arealtered, comprising a compound of formula I, II or III, as definedabove, labeled with a marker.

Finally, this invention relates to a pharmaceutical compositioncomprising:

(i) an effective amount of a compound of formula I, II or III, asdescribed above; and

(ii) a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effect of2-(phosphonomethyl)pentanedioic acid (“Compound C”) on TGF-β1concentrations in ischemic cell cultures.

FIG. 2 is a bar graph showing the effect of Compound C on TGF-β2concentrations in ischemic cell cultures.

FIG. 3 is a bar graph showing the reversal of the neuroprotective effectof Compound C by TGF-β neutralizing antibodies in ischemic cellcultures.

FIG. 4 is a bar graph showing the non-reversal of the neuroprotectiveeffect of Compound C by FGF neutralizing antibodies in ischemic cellcultures.

FIG. 5 is a bar graph showing the reversal of the neuroprotective effectof Compound C by TGF-β neutralizing antibodies in rats subjected tomiddle cerebral artery occlusion (“MCAO”).

FIG. 6 is a bar graph showing the effect of Compound C on TGF-β1 levelsduring occlusion and reperfusion in rats subjected to MCAO.

FIG. 7A is a bar graph plotting the withdrawal latency difference scoresof non-diabetic rats and STZ-diabetic rats treated with a vehicle or2-[[2,3,4,5,6-pentafluorobenzyl) hydroxyphosphinyl]methyl]pentanedioicacid (“Compound A”), against the days following administration withstreptozotocin (“STZ”).

FIG. 7B is a bar graph plotting the withdrawal latency difference scoresof non-diabetic rats and STZ-diabetic rats treated with a vehicle or2-(2-sulfanylethyl)pentanedioic acid (“Compound D”), against the daysfollowing administration with STZ.

FIG. 8 is a bar graph plotting the withdrawal latency difference scoresof normal (unoperated) rats and chronic constrictive injury-induced ratstreated with a vehicle or Compound C, against the days followingsurgery.

FIG. 9A is a bar graph plotting the motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound A, against the weeks post STZ.

FIG. 9B is a bar graph plotting the sensory nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound A, against the weeks post STZ.

FIG. 10A is a bar graph plotting the motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound D, against the weeks post STZ.

FIG. 10B is a bar graph plotting the sensory nerve conduction velocityof non-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound D, against the weeks post STZ.

FIG. 11 is a graph plotting the withdrawal latency of non-diabetic ratsand BB/W diabetic rats treated with a vehicle, Compound D or Compound A,against the weeks of treatment.

FIG. 12 is a graph plotting the nerve conduction velocity ofnon-diabetic rats and BB/W diabetic rats treated with a vehicle,Compound D or Compound A, against the weeks of treatment.

FIG. 13 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 10 mg/kg3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)-benzenepropanoicacid(“Compound 49”), against the days following treatment.

FIG. 14 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 1 mg/kg Compound 49, against the days following treatment.

FIG. 15 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 0.1 mg/kg Compound 49, against the days following treatment.

FIG. 16 is a bar graph plotting the withdrawal latency of chronicconstrictive injury-induced rats treated with a vehicle or 10, 1 or 0.1mg/kg Compound 49, against the days following treatment.

FIG. 17 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 50 mg/kg 3-carboxy-alpha-(3-mercaptopropyl)-benzenepropanoicacid (“Compound 5”), against the days following treatment.

FIG. 18 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 30 mg/kg Compound 5, against the days following treatment.

FIG. 19 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 10 mg/kg Compound 5, against the days following treatment.

FIG. 20 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 3 mg/kg Compound 5, against the days following treatment.

FIG. 21 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 1 mg/kg Compound 5, against the days following treatment.

FIG. 22 is a bar graph plotting the withdrawal latency differences inscores of chronic constrictive injury-induced rats treated with avehicle or 0.3 mg/kg Compound 5, against the days following treatment.

FIG. 23 is a bar graph plotting the withdrawal latency of chronicconstrictive injury-induced rats treated with a vehicle or 10, 30 or 50mg/kg Compound 5, against the days following treatment.

FIG. 24 is a bar graph plotting the percent of transgenic mice at 210days of age that exhibited limb shaking after treatment with2-(3-sulfanylpropyl)pentanedioic acid (“Compound B”) or a vehicle.

FIG. 25 is a bar graph plotting the gait, measured on an arbitrary scaleranging from 0 to 3, of transgenic mice at 210 days of age aftertreatment with Compound B or a vehicle.

FIG. 26 is a bar graph plotting hind limbs dragging, measured on anarbitrary scale ranging from 0 to 3, of transgenic mice at 210 days ofage after treatment with Compound B or a vehicle.

FIG. 27 is a bar graph plotting the crossing of limbs, measured on anarbitrary scale ranging from 0 to 3, of transgenic mice at 210 days ofage after treatment with Compound B or a vehicle.

FIG. 28 is a bar graph plotting the righting reflex of transgenic mice,measured by the time (seconds) it took the mice to right themselves whenplaced on their sides, at 210 days of age after treatment with CompoundB or a vehicle.

FIG. 29 is a graph plotting the percent of transgenic mice treated withCompound B or a vehicle that died against the age of the mice (days).

FIG. 30 is a Kaplan-Meier survival graph plotting the percent oftransgenic mice treated with Compound B or a vehicle that survivedagainst the number of days that the mice were on study therapy.

FIG. 31 is a bar graph plotting the withdrawal latency difference scoresof non-diabetic rats and STZ-diabetic rats treated with a vehicle,Compound D or 3-carboxy-alpha-(3-mercaptopropyl)benzenepropanoic acid(“Compound E”), against the weeks of treatment.

FIG. 32 is a bar graph plotting motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle, CompoundD or Compound E, against the weeks of treatment.

FIG. 33 is a bar graph plotting sensory nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle, CompoundD or Compound E, against the weeks of treatment, where treatment started5 weeks post STZ.

FIG. 34 is a bar graph plotting the withdrawal latency difference scoresof non-diabetic rats and STZ-diabetic rats treated with a vehicle orlower doses of Compound D (1 and 3 mg/kg), against the weeks oftreatment, where treatment started 7 weeks post STZ.

FIG. 35 is a bar graph plotting motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle or lowerdoses of Compound D (1 and 3 mg/kg), against the weeks of treatment,where treatment started 7 weeks post STZ.

FIG. 36 is a bar graph plotting sensory nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle or lowerdoses of Compound D (1 and 3 mg/kg), against the weeks of treatment,where treatment started 7 weeks post STZ.

FIG. 37 are bar graphs plotting sensory nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound D at 35 days and 60 days after treatment, where treatmentstarted 60 days post STZ.

FIG. 38 are bar graphs plotting motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound D at 35 days after treatment, where treatment started 60 dayspost STZ.

FIG. 39 is a graph plotting sensory nerve conduction velocity ofnon-diabetic and STZ-diabetic rats treated with a vehicle or Compound D,against the days post STZ, where treatment started 90 days post STZ.

FIG. 40 is a bar graph plotting motor and sensory nerve conductionvelocities of non-diabetic mice and db/db diabetic mice before treatmentwith a NAALADase inhibitor.

FIG. 41 is a bar graph plotting motor and sensory nerve conductionvelocities of non-diabetic mice and db/db diabetic mice after treatmentwith3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)benzenepropanoicacid (“Compound F”).

FIG. 42 is bar graph comparing the rotarod performance of transgenic HDmice and normal non-HD mice treated with Compound B, and transgenic HDmice and normal non-HD mice treated with a vehicle.

FIG. 43 is a bar graph comparing the total distance traveled bytransgenic HD mice and normal non-HD mice treated with Compound B, andtransgenic HD mice and normal non-HD mice treated with a vehicle.

FIG. 44 is a graph plotting the survival time of transgenic D micetreated with Compound B or a vehicle.

FIG. 45 is a graph plotting the survival time of male transgenic HD micetreated with Compound B or a vehicle.

FIG. 46 is a graph plotting the survival time of female transgenic HDmice treated with Compound B or a vehicle.

DETAILED DESCRIPTION

Definitions

“Compound A” refers to2-[[2,3,4,5,6-pentafluorobenzyl)hydroxyphosphinyl]methyl]pentanedioicacid.

“Compound B” refers to 2-(3-sulfanylpropyl)pentanedioic acid.

“Compound C” refers to 2-(phosphonomethyl)pentanedioic acid (PMPA).

“Compound D” refers to 2-(2-sulfanylethyl)pentanedioic acid.

“Compound E” refers to3-carboxy-alpha-(3-mercaptopropyl)benzenepropanoic acid.

“Compound F” refers to3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)benzenepropanoicacid.

“Alkyl” refers to a branched or unbranched saturated hydrocarbon chaincomprising a designated number of carbon atoms. For example, C₁-C₉ alkylis a straight or branched hydrocarbon chain containing 1 to 9 carbonatoms, and includes but is not limited to substituents such as methyl,ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl,n-hexyl, and the like, unless otherwise indicated.

“Alkenyl” refers to a branched or unbranched unsaturated hydrocarbonchain comprising a designated number of carbon atoms. For example, C₂-C₉alkenyl is a straight or branched hydrocarbon chain containing 2 to 9carbon atoms having at least one double bond, and includes but is notlimited to substituents such as ethenyl, propenyl, iso-propenyl,butenyl, iso-butenyl, tert-butenyl, n-pentenyl, n-hexenyl, and the like,unless otherwise indicated.

“Alkoxy” refers to the group —OR wherein R is alkyl as herein defined.Preferably, R is a branched or unbranched saturated hydrocarbon chaincontaining 1 to 9 carbon atoms.

“Carbocycle” refers to a hydrocarbon, cyclic moiety having one or moreclosed ring(s) that is/are alicyclic, aromatic, fused and/or bridged.Examples include cyclopropane, cyclobutane, cyclopentane, cyclohexane,cycloheptane, cyclopentene, cyclohexene, cycloheptene, cycloctene,benzyl, naphthene, anthracene, phenanthracene, biphenyl and pyrene.

“Aryl” refers to an aromatic, hydrocarbon cyclic moiety having one ormore closed rings Examples include, without limitation, phenyl, benzyl,naphthyl, anthracenyl, phenanthracenyl, biphenyl and pyrenyl.

“Heterocycle” refers to a cyclic moiety having one or more closed ringsthat is/are alicyclic, aromatic, fused and/or bridged, with one or moreheteroatoms (for example, sulfur, nitrogen or oxygen) in at least one ofthe rings. Examples include, without limitation, pyrrolidine, pyrrole,thiazole, thiophene, piperidine, pyridine, isoxazolidine and isoxazole.

“Heteroaryl” refers to an aromatic, cyclic moietyhaving one or moreclosed rings with one or more heteroatoms (for example, sulfur, nitrogenor oxygen) in at least one of the rings. Examples include, withoutlimitation, pyrrole, thiophene, pyridine and isoxazole.

“Derivative” refers to a substance produced from another substanceeither directly or by modification or partial substitution.

“Effective amount” refers to the amount required to produce the desiredeffect, for example, to inhibit NAALADase enzyme activity and/orangiogenesis, to effect neuronal activity or TGF-β activity, and/or totreat glutamate abnormality, compulsive disorder, prostate disease,cancer or glaucoma.

“Electromagnetic radiation” includes without limitation radiation havingthe wavelength of 10⁻²⁰ to 10⁰ meters. Examples include, withoutlimitation, gamma radiation (10⁻²⁰ to 10⁻¹³ m), X-ray radiation (10⁻¹¹to 10⁻⁹ m), ultraviolet light (10 nm to 400 nm), visible light (400 nmto 700 nm) infrared radiation (700 nm to 1.0 mm) and microwave radiation(1 mm to 30 cm).

“Halo” refers to at least one fluoro, chloro, bromo or iodo moiety.

“Isosteres” refer to elements, functional groups, substitutents,molecules or ions having different molecular formulae but exhibitingsimilar or identical physical properties. For example, tetrazole is anisostere of carboxylic acid because it mimics the properties ofcarboxylic acid even though they both have different molecular formulae.Typically, two isosteric molecules have similar or identical volumes andshapes. Ideally, isosteric compounds should be isomorphic and able toco-crystallize. Other physical properties that isosteric compoundsusually share include boiling point, density, viscosity and thermalconductivity. However, certain properties are usually different: dipolarmoments, polarity, polarization, size and shape since the externalorbitals may be hybridized differently. The term “isosteres” encompasses“bioisosteres”.

“Bioisosteres” are isosteres that, in addition to their physicalsimilarities, share some common biological properties. Typically,bioisosteres interact with the same recognition site or produce broadlysimilar biological effects.

“Carboxylic acid isosteres” include without limitation directderivatives such as hydroxamic acids, acyl-cyanamides andacylsulfonamides; planar acidic heterocycles such as tetrazoles,mercaptoazoles, sulfinylazoles, sulfonylazoles, isoxazoles,isothiazoles, hydroxythiadiazoles and hydroxychromes; and nonplanarsulfur- or phosphorus-derived acidic functions such as phosphinates,phosphonates, phosphonamides, sulphonates, sulphonamides, andacylsulphonamides.

“Metabolite” refers to a substance produced by metabolism or by ametabolic process.

“NAAG” refers to N-acetyl-aspartyl-glutamate, an important peptidecomponent of the brain, with levelscomparable to the major inhibitorneurotransmitter gamma-aminobutyric acid (“GABA”). NAAG isneuron-specific, present in synaptic vesicles and released upon neuronalstimulation in several systems presumed to be glutamatergic. Studiessuggest that NAAG may function as a neurotransmitter and/orneuromodulator in the central nervous system, or as a precursor of theneurotransmitter glutamate. In addition, NAAG is an agonist at group IImetabotropic glutamate receptors, specifically mGluR3 receptors; whenattached to a moiety capable of inhibiting NAALADase, it is expectedthat metabotropic glutamate receptor ligands will provide potent andspecific NAALADase inhibitors.

“NAALADase” refers to N-acetylated α-linked acidic dipeptidase, amembrane bound metallopeptidase which catabolizes NAAG toN-acetylaspartate (“NAA”) and glutamate (“GLU”):

Catabolism of NAAG by NAALADase

NAALADase has been assigned to the M28 peptidase family and is alsocalled PSMA or human GCP II, EC number 3.4.17.21. It is believed thatNAALADase is a co-catalytic zinc/zinc metallopeptidase. NAALADase showsa high affinity for NAAG with a Km of 540 nM. If NAAG is a bioactivepeptide, then NAALADase may serve to inactivate NAAG's synaptic action.Alternatively, if NAAG functions as a precursor for glutamate, theprimary function of NAALADase may be to regulate synaptic glutamateavailability.

“Pharmaceutically acceptable carrier” refers to any carrier, diluent,excipient, wetting agent, buffering agent, suspending agent, lubricatingagent, adjuvant, vehicle, delivery system, emulsifier, disintegrant,absorbent, preservative, surfactant, colorant, flavorant, or sweetener,preferably non-toxic, that would be suitable for use in a pharmaceuticalcomposition.

“Pharmaceutically acceptable equivalent” includes, without limitation,pharmaceutically acceptable salts, hydrates, metabolites, prodrugs andisosteres. Many pharmaceutically acceptable equivalents are expected tohave the same or similar in vitro or in vivo activity as the compoundsof the invention.

“Pharmaceutically acceptable salt” refers to a salt of the inventivecompounds which possesses the desired pharmacological activity and whichis neither biologically nor otherwise undesirable. The salt can beformed with acids that include, without limitation, acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycero-phosphate, hemisulfate, heptanoate, hexanoate, hydrochloridehydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,thiocyanate, tosylate and undecanoate. Examples of a base salt includeammonium salts, alkali metal salts such as sodium and potassium salts,alkaline earth metal salts such as calcium and magnesium salts, saltswith organic bases such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine andlysine. Basic nitrogen-containing groups can be quarternized with agentsincluding lower alkyl halides such as methyl, ethyl, propyl and butylchlorides, bromides and iodides; dialkyl sulfates such as dimethyl,diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides; andaralkyl halides such as benzyl and phenethyl bromides.

“Prodrug” refers to a derivative of the inventive compounds thatundergoes biotransformation, such as metabolism, before exhibiting itspharmacological effect(s) The prodrug is formulated with theobjective(s) of improved chemical stability, improved patient acceptanceand compliance, improved bioavailability, prolonged duration of action,improved organ selectivity, improved formulation (e.g., increasedhydrosolubility), and/or decreased side effects (e.g., toxicity). Theprodrug can be readily prepared from the inventive compounds usingmethods known in the art, such as those described by Burger's MedicinalChemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172-178, 949-982(1995).

“Radiosensitizer” refers to a low molecular weight compound administeredto animals in therapeutically effective amounts to promote the treatmentof diseases that are treatable with electromagnetic radiation. Diseasesthat are treatable with electromagnetic radiation include, withoutlimitation, neoplastic diseases, benign and malignant tumors, andcancerous cells. Electromagnetic radiation treatment of other diseasesnot listed herein are also contemplated by this invention.

“Inhibition,” in the context of enzymes, refers to reversible enzymeinhibition such as competitive, uncompetitive and non-competitiveinhibition. Competitive, uncompetitive and non-competitive inhibitioncan be distinguished by the effects of an inhibitor on the reactionkinetics of an enzyme. Competitive inhibition occurs when the inhibitorcombines reversibly with the enzyme in such a way that it competes witha normal substrate for binding at the active site. The affinity betweenthe inhibitor and the enzyme may be measured by the inhibitor constant,K_(i), which is defined as:$K_{i} = \frac{\lbrack E\rbrack \lbrack I\rbrack}{\lbrack{EI}\rbrack}$

wherein [E] is the concentration of the enzyme, [I] is the concentrationof the inhibitor, and [EI] is the concentration of the enzyme-inhibitorcomplex formed by the reaction of the enzyme with the inhibitor. Unlessotherwise specified, K_(i) as used herein refers to the affinity betweenthe inventive compounds and NAALADase. “IC₅₀” is a related term used todefine the concentration or amount of a compound that is required tocause a 50% inhibition of the target enzyme.

“NAALADase inhibitor” refers to any compound that inhibits NAALADaseenzyme activity. Preferably, a NAALADase inhibitor exhibits a K_(i) ofless than 100 μM, more preferably less than 10 μM, and even morepreferably less than 1 μM, as determined using any appropriate assayknown in the art.

“Isomers” refer to compounds having the same number and kind of atoms,and hence the same molecular weight, but differing in respect to thearrangement or configuration of the atoms.

“Optical isomers” refer to enantiomers or diastereoisomers.

“Stereoisomers” are isomers that differ only in the arrangement of theatoms in space.

“Diastereoisomers” are stereoisomers that are not mirror images of eachother. Diastereoisomers occur in compounds having two or more asymmetriccarbon atoms; thus, such compounds have 2^(n) optical isomers, where nis the number of asymmetric carbon atoms.

“Enantiomers” are a pair of stereoisomers that are non-superimposablemirror images of each other. Enantiomers result from the presence of oneor more asymmetric carbon atoms in the compound (e.g., glyceraldehyde,lactic acid, sugars, tartaric acid, amino acids).

“Enantiomer-enriched” refers to a mixture in which one enantiomerpredominates.

“Racemic mixture means a mixture containing equal amounts of individualenantiomers.

“Non-racemic mixture” is a mixture containing unequal amounts ofenantiomers.

“Angiogenesis” refers to the process whereby new capillaries are formed.“Inhibition” of angiogenesis may be measured by many parameters inaccordance with this invention and, for instance, may be assessed bydelayed appearance of neovascular structures, slowed development ofneovascular structures, decreased occurrence of neovascular structures,slowed or decreased severity of angiogenesis-dependent disease effects,arrested angiogenic growth, or regression of previous angiogenic growth.In the extreme, complete inhibition is referred to herein as prevention.In relation to angiogenesis or angiogenic growth, “prevention” refers tono substantial angiogenesis or angiogenic growth if none had previouslyoccurred, or no substantial further angiogenesis or angiogenic growth ifgrowth had previously occurred.

“Angiogenesis-dependent disease” includes, without limitation,rheumatoid arthritis, cardiovascular diseases, neovascular diseases ofthe eye, peripheral vascular disorders, dermatologic ulcers andcancerous tumor growth, invasion and metastasis.

“Animal” refers to a living organism having sensation and the power ofvoluntary movement, and which requires for its existence oxygen andorganic food. Examples include, without limitation, members of thehuman, equine, porcine, bovine, murine, canine, or feline species. Inthe case of a human, an “animal” may also be referred to as a “patient”.

“Mammal” refers to a warm-blooded vertebrate animal.

“Anxiety” includes without limitation the unpleasant emotion stateconsisting of psychophysiological responses to anticipation of unreal orimagined danger, ostensibly resulting from unrecognized intrapsychicconflict. Physiological concomitants include increased heart rate,altered respiration rate, sweating, trembling, weakness, and fatigue;psychological concomitants include feelings of impending danger,powerlessness, apprehension, and tension. Dorland's Illustrated MedicalDictionary, W. B. Saunders Co., 27th ed. (1988).

“Anxiety Disorder” includes without limitation mental disorders in whichanxiety and avoidance behavior predominate. Dorland's IllustratedMedical Dictionary, W. B. Saunders Co., 27th ed. (1988). Examplesinclude without limitation panic attack, agoraphobia, panic disorder,acute stress disorder, chronic stress disorder, specific phobia, simplephobia, social phobia, substance induced anxiety disorder, organicanxiety disorder, obsessive compulsive disorder, post-traumatic stressdisorder, generalized anxiety disorder, and anxiety disorder NOS. Otheranxiety disorders are characterized in Diagnostic and Statistical Manualof Mental Disorders (American Psychiatric Association 4th ed. 1994).

“Attention Deficit Disorder” or “ADD” refers to a disorder characterizedby developmentally inappropriate inattention and impulsiveness, with orwithout hyperactivity. Inattention means a failure to finish tasksstarted, easily distracted, seeming lack of attention, and difficultyconcentrating on tasks requiring sustained attention. Impulsivenessmeans acting before thinking, difficulty taking turns, problemsorganizing work, and constant shifting from one activity to another.Hyperactivity means difficulty staying seated and sitting still, andrunning or climbing excessively.

“Cancer” includes, without limitation, ACTH-producing tumors, acutelymphocytic leukemia, acute nonlymphocytic leukemia, cancer of theadrenal cortex, bladder cancer, brain cancer, breast cancer, cervixcancer, chronic lymphocytic leukemia, chronic myelocytic leukemia,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cellleukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma,kidney cancer, liver cancer, lung cancer (small and/or non-small cell),malignant peritoneal effusion, malignant pleural effusion, melanoma,mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma,osteosarcoma, ovary cancer, ovary (germ cell) cancer, pancreatic cancer,penis cancer, prostate cancer, retinoblastoma, skin cancer, soft-tissuesarcoma, squamous cell carcinomas, stomach cancer, testicular cancer,thyroid cancer, trophoblastic neoplasms, cancer of the uterus, vaginalcancer, cancer of the vulva, and Wilm's tumor.

“Compulsive disorder” refers to any disorder characterized byirresistible impulsive behavior. Examples of compulsive disordersinclude without limitation substance dependence, eating disorders,pathological gambling, Attention Deficit Disorder (“ADD”), andTourette's syndrome.

“Demyelinating disease” refers to any disease involving damage to orremoval of the myelin sheath naturally surrounding nerve tissue, such asthat defined in U.S. Pat. No. 5,859,046 and International PublicationNo. WO 98/03178, herein incorporated by reference. Examples includewithout limitation peripheral demyelinating diseases (such asGuillain-Barré syndrome, peripheral neuropathies and Charcot-Marie Toothdisease) and central demyelinating diseases (such as multiplesclerosis).

“Disease” refers to any deviation from or interruption of the normalstructure or function of any part, organ or system (or combinations) ofthe body that is manifested by a characteristic set of symptoms andsigns and whose etiology, pathology, and prognosis may be known orunknown. Dorland's Illustrated Medical Dictionary, (W. B. Saunders Co.27th ed. 1988).

“Disorder” refers to any derangement or abnormality offunction; a morbidphysical or mental state. Dorland's Illustrated Medical Dictionary, (W.B. Saunders Co. 27th ed. 1988).

“Drug dependence” refers to a psychologic addiction or a physicaltolerance to a drug. Tolerance means a need to increase the doseprogressively in order to produce the effect originally achieved bysmaller amounts.

“Eating disorder” refers to compulsive overeating, obesity or severeobesity. Obesity means body weight of 20% over standard height-weighttables. Severe obesity means over 100% overweight.

“Glaucoma” includes without limitation chronic (idiopathic) open-angleglaucomas (e.g., high-pressure, normal-pressure); pupillary blockglaucomas (e.g., acute angle-closure, subacute angle-closure, chronicangle-closure, combined-mechanism); developmental glaucomas (e.g.,congenital (infantile), juvenile, Anxenfeld-Rieger syndrome, Peters'anomaly, Aniridia); glaucomas associated with other ocular disorders(e.g., glaucomas associated with disorders of the corneal endothelium,iris, ciliary body, lens, retina, choroid and vitreous); glaucomasassociated with elevated episcleral venous pressure (e.g., systemicdiseases with associated elevated intraocular pressure and glaucoma,corticosteroid-induced glaucoma); glaucomas associated with inflammationand trauma (e.g., glaucomas associated with keratitis, episcleritis,scleritis, uveitis, ocular trauma and hemorrhage); glaucomas followingintraocular surgery (e.g., ciliary block (malignant) glaucoma, glaucomasin aphakia and pseudophakia, glaucomas associated with corneal surgery,glaucomas associated with vitreoretinal surgery).

“Glutamate abnormality” refers to any disease, disorder, or condition inwhich glutamate is implicated, including pathological conditionsinvolving elevated levels of glutamate. Examples of glutamateabnormalities include,without limitation, compulsive disorder, spinalcord injury, epilepsy, stroke, ischemia, demyelinating disease,Alzheimer's disease, Parkinson's disease, ALS, Huntington's disease,schizophrenia, pain, peripheral neuropathy (including but not limited todiabetic neuropathy), traumatic brain injury, neuronal insult,inflammatory disease, anxiety, anxiety disorder, memory impairment andglaucoma.

“Ischemia” refers to localized tissue anemia due to obstruction of theinflow of arterial blood. Global ischemia occurs when blood flow ceasesfor a period of time, as may result from cardiac arrest. Focal ischemiaoccurs when a portion of the body, such as the brain, is deprived of itsnormal blood supply, such as may result from thromboembolytic occlusionof a cerebral vessel, traumatic head injury, edema or brain tumor. Evenif transient, both global and focal ischemia can produce widespreadneuronal damage. Although nerve tissue damage occurs over hours or evendays following the onset of ischemia, some permanent nerve tissue damagemay develop in the initial minutes following cessation of blood flow tothe brain. Much of this damage is attributed to glutamate toxicity andsecondary consequences of reperfusion of the tissue, such as the releaseof vasoactive products by damaged endothelium, and the release ofcytotoxic products, such as free radicals and leukotrienes, by thedamaged tissue.

“Memory impairment” refers to a diminished mental registration,retention or recall of past experiences, knowledge, ideas, sensations,thoughts or impressions. Memory impairment may affect short andlong-term information retention, facility with spatial relationships,memory (rehearsal) strategies, and verbal retrieval and production.Common causes of memory impairment are age, severe head trauma, brainanoxia or ischemia, alcoholic-nutritional diseases, drug intoxicationsand neurodegenerative diseases. For example, memory impairment is acommon feature of neurodegenerative diseases such as Alzheimer's diseaseand senile dementia of the Alzheimer type. Memory impairment also occurswith other kinds of dementia such as multi-infarct dementia, a seniledementia caused by cerebrovascular deficiency, and the Lewy-body variantof Alzheimer's disease with or without association with Parkinson'sdisease. Creutzfeldt-Jakob disease is a rare dementia with which memoryimpairment is associated. It is a spongiform encephalopathy caused bythe prion protein; it may be transmitted from other sufferers or mayarise from gene mutations. Loss of memory is also a common feature ofbrain-damaged patients. Brain damage may occur, for example, after aclassical stroke or as a result of an anaesthetic accident, head trauma,hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin(B₁, thiamine and B₁₂) deficiency, or excessive alcohol use. Korsakoff'samnesic psychosis is a rare disorder characterized by profound memoryloss and confabulation, whereby the patient invents stories to concealhis or her memory loss. It is frequently associated with excessivealcohol intake. Memory impairment may furthermore be age-associated; theability to recall information such as names, places and words seems todecrease with increasing age. Transient memory loss may also occur inpatients, suffering from a major depressive disorder, afterelectro-convulsive therapy.

“Mental disorder” refers to any clinically significant behavioral orpsychological syndrome characterized by the presence of distressingsymptoms or significant impairment of functioning. Mental disorders areassumed to result from some psychological or organic dysfunction of theindividual; the concept does not include disturbances that areessentially conflicts between the individual and society (socialdeviance).

“Metastasis” refers to “[t]he ability of cells of a cancer todisseminate and form new foci of growth at noncontiguous sites (i.e., toform metastases).” See Hill, R. P, “Metastasis”, The Basic Science ofOncology, Tannock et al., Eds., McGraw-Hill, N.Y., pp. 178-195 (1992),herein incorporated by reference. “The transition from in situ tumorgrowth to metastatic disease is defined by the ability of tumor cells ofthe primary site to invade local tissues and to cross tissue barriers .. . . To initiate the metastatic process, carcinoma cells must firstpenetrate the epithelial basement membrane and then invade theinterstitial stroma. . . . For distant metastases, intravasationrequires tumor cell invasion of the subendothelial basement membranethat must also be negotiated during tumor cell extravasation . . . Thedevelopment of malignancy is also associated with tumor-inducedangiogenesis [which] not only allows for expansion of the primarytumors, but also permits easy access to the vascular compartment due todefects in the basement membranes of newly formed vessels.” SeeAznavoorian et al., Cancer (1993) 71:1368-1383, herein incorporated byreference.

“Nervous insult” refers to any damage to nervous tissue and anydisability or death resulting therefrom. The cause of nervous insult maybe metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, andincludes without limitation ischemia, hypoxia, cerebrovascular accident,trauma, surgery, pressure, mass effect, hemorrhage, radiation,vasospasm, neurodegenerative disease, neurodegenerative process,infection, Parkinson's disease, ALS, myelination/demyelinationprocesses, epilepsy, cognitive disorder, glutamate abnormality andsecondary effects thereof.

“Nervous tissue” refers to the various components that make up thenervous system, including without limitation neurons, neural supportcells, glia, Schwann cells, vasculature contained within and supplyingthese structures, the central nervous system, the brain, the brain stem,the spinal cord, the junction of the central nervous system with theperipheral nervous system, the peripheral nervous system and alliedstructures.

“Neuropathy” refers to any disease or malfunction of the nerves.Neuropathy includes, without limitation, peripheral neuropathy, diabeticneuropathy, autonomic neuropathy and mononeuropathy. Peripheralneuropathy may be idiopathic or induced by any causes including diseases(for example, amyloidosis, alcoholism, HIV, syphilis, virus, autoimmunedisorder, cancer, porphyria, arachnoiditis, post herpetic neuralgia,Guillain-Barré syndrome, diabetes including type I and type IIdiabetes), chemicals (for example, toxins, lead, dapsone, vitamins,paclitaxel chemotherapy, HAART therapy) and physical injuries to aparticular nerve or nerve plexus (for example, trauma, compression,constriction).

“Neuroprotective” refers to the effect of reducing, arresting orameliorating nervous insult, and protecting, resuscitating or revivingnervous tissue that has suffered nervous insult.

“Pain” refers to localized sensations of discomfort, distress or agony,resulting from the stimulation of specialized nerve endings. It servesas a protective mechanism insofar as it induces the sufferer to removeor withdraw from the source. Dorland's Illustrated Medical Dictionary,(W. B. Saunders Co. 27th ed. 1988). Examples of pain include, withoutlimitation, acute, chronic, cancer, burn, incisional, inflammatory,neuropathic and back pain.

“Neuropathic pain” refers to a condition of pain associated with a nerveinjury. Depending on the particular syndrome, the pain may be due toalterations of the brain or spinal cord or may be due to abnormalitiesin the nerve itself. Neuropathic pain may be idiopathic or induced byany causes including diseases (for example, amyloidosis, alcoholism,HIV, syphilis, virus, autoimmune disorder, cancer, porphyria,arachnoiditis, post herpetic neuralgia, Guillain-Barré syndrome,diabetes including type I and type II diabetes), chemicals (for example,toxins, lead, dapsone, vitamins, paclitaxel chemotherapy, HAART therapy)and physical injuries to a particular nerve or nerve plexus (forexample, trauma, compression, constriction).

“Pathological gambling” refers to a condition characterized by apreoccupation with gambling. Similar to psychoactive substance abuse,its effects include development of tolerance with a need to gambleprogressively larger amounts of money, withdrawal symptoms, andcontinued gambling despite severe negative effects on family andoccupation.

“Prostate disease” refers to any disease affecting the prostate.Examples of prostate disease include without limitation prostate cancersuch as adenocarcinoma and metastatic cancers of the prostate; andconditions characterized by abnormal growth of prostatic epithelialcells such as benign prostatic hyperplasia.

“Schizophrenia” refers to a mental disorder or group of mental disorderscharacterized by disturbances in form and content of thought (looseningof associations, delusions, hallucinations), mood (blunted, flattened,inappropriate affect), sense of self and relationship to the externalworld (loss of ego boundaries, dereistic thinking, and autisticwithdrawal), and behavior (bizarre, apparently purposeless, andstereotyped activity or inactivity). Examples of schizophrenia include,without limitation, acute, ambulatory, borderline, catatonic, childhood,disorganized, hebephrenic, latent, nuclear, paranoid, paraphrenic,prepsychotic, process, pseudoneurotic, pseudopsychopathic, reactive,residual, schizo-affective and undifferentiated schizophrenia. Dorland'sIllustrated Medical Dictionary, (W. B. Saunders Co. 27th ed. 1988).

“TGF-β” refers to transforming growth factor beta. TGF-β is recognizedas a prototype of multifunctional growth factors. It regulates variouscell and tissue functions, including cell growth and differentiation,angiogenesis, wound healing, immune function, extracellular matrixproduction, cell chemotaxis, apoptosis and hematopoiesis.

“TGF-β abnormality” refers to any disease, disorder or condition inwhich TGF-β is implicated, including diseases disorders and conditionscharacterized by an abnormal level of TGF-β.

“Abnormal level of TGF-β” refers to a measurable variance from normallevels of TGF-β, as determined by one of ordinary skill in the art usingknown techniques.

“Therapeutic window of opportunity” or “window” refers, in relation tostroke, to the maximal delay between the onset of stroke and theinitiation of efficacious therapy.

“Tourette's syndrome” refers to an autosomal multiple tic disordercharacterized by compulsive swearing, multiple muscle tics and loudnoises. Tics are brief, rapid, involuntary movements that can be simpleor complex; they are stereotyped and repetitive, but not rhythmic.Simple tics, such as eye blinking, often begin as nervous mannerisms.Complex tics often resemble fragments of normal behavior.

Unless otherwise defined in conjunction with specific diseases ordisorders, “treating” refers to:

(i) preventing a disease, disorder or condition from occurring in ananimal that may be predisposed to the disease, disorder and/or conditionbut has not yet been diagnosed as having it;

(ii) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and/or

(iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

“Treating ALS” refers to:

(i) preventing ALS from occurring in an animal that may be predisposedto ALS but has not yet been diagnosed as having it;

(ii) inhibiting ALS, i.e. arresting its development;

(iii) relieving ALS, i.e. causing regression of the disease, disorderand/or condition;

(iv) delaying onset of ALS or ALS symptom(s);

(v) slowing progression of ALS or ALS symptom(s);

(vi) prolonging survival of an animal suffering from ALS; and/or

(vii) attenuating ALS symptom(s).

“Treating Huntington's disease” refers to:

(i) preventing Huntington's disease from occurring in an animal that maybe predisposed to Huntington's disease but has not yet been diagnosed ashaving it;

(ii) inhibiting or slowing Huntington's disease, e.g. arresting itsdevelopment;

(iii) relieving Huntington's disease, e.g. causing its regression;

(iv) improving motor coordination in an animal having Huntington'sdisease; and/or

(v) prolonging the survival of an animal having Huntington's disease.

“Treating substance dependence” refers to preventing relapse; reducingcraving; suppressing tolerance; preventing, inhibiting and/or relievingwithdrawal; attenuating sensitization; preventing, inhibiting (i.e.arresting development of) and/or relieving (i.e. causing regression of)substance-induced neurotoxicity; and/or preventing, inhibiting and/orrelieving fetal alcohol syndrome.

“Craving” refers to a strong desire for a substance and/or a compellingurge and/or an irresistible impulse to use a substance.

“Dependence” refers to a maladaptive pattern of substance use, leadingto clinically significant impairment or distress. Dependence istypically characterized by tolerance and/or withdrawal. Substances forwhich dependence may be developed include, without limitation,depressants (opioids, synthetic narcotics, barbiturates, glutethimide,methyprylon, ethchlorvynol, methaqualone, alcohol); anxiolytics(diazepam, chlordiazepoxide, alprazolam, oxazepam, temazepam);stimulants (amphetamine, methamphetamine, cocaine); and hallucinogens(LSD, mescaline, peyote, marijuana).

“Relapse” refers to a return to substance use after a period ofabstinence, often accompanied by reinstatement.

“Reinstatement” refers to a return to a preexisting level of use anddependence in a person who has resumed substance use following a periodof abstinence.

“Sensitization” refers to a condition in which the response to asubstance increases with repeated use.

“Tolerance” refers to an acquired reaction to a substance characterizedby diminished effect with continued use of the same dose and/or a needfor increased doses to achieve intoxication or desired effect previouslyachieved by lower doses. Both physiological and psychosocial factors maycontribute to the development of tolerance. With respect tophysiological tolerance, metabolic and/or functional tolerance maydevelop. By increasing the rate of metabolism of the substance, the bodymay be able to eliminate the substance more readily. Functionaltolerance is defined as a decrease in sensitivity of the central nervoussystem to the substance.

“Withdrawal” refers to a syndrome characterized by untoward physicalchanges that occur following cessationof or reduction in substance use,or administration of a pharmacologic antagonist.

One of ordinary skill in the art will recognize that there arealternative nomenclatures, nosologies and classification systems for thediseases, disorders and conditions defined above, and that such systemsevolve with medical scientific progress.

Unless the context clearly dictates otherwise, the definitions ofsingular terms may be extrapolated to apply to their plural counterpartsas they appear in the application; likewise, the definitions of pluralterms may be extrapolated to apply to their singular counterparts asthey appear in the application.

COMPOUNDS OF THE INVENTION

This invention relates to compounds of formula I

or a pharmaceutically acceptable equivalent of said compound, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen or C₁-C₃alkyl;

A¹, A², A³ and A⁴ are independently hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy,halo, nitro, phenyl, phenoxy, benzyl, benzyloxy or —COOH, or anyadjacent two of A², A³ and A⁴ form with the benzene ring a fused 5- or6-membered carbocyclic or heterocyclic aromatic ring, said heterocyclicaromatic ring containing 1 or 2 oxygen, nitrogen and/or sulfurheteroatom(s).

In one embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independentlyhydrogen or methyl; and A¹, A², A³ and A⁴ are independently hydrogen,C₁-C₄ alkyl, C₁-C₂ alkoxy, halo, nitro, phenyl, phenoxy, benzyloxy,nitro or —COOH.

In another embodiment, any adjacent two of A², A³ and A⁴ form with thebenzene ring a fused 5- or 6-membered carbocyclic or heterocyclicaromatic ring, said heterocyclic aromatic ring containing 1 or 2 oxygen,nitrogen and/or sulfur heteroatom(s).

This invention further relates to a compound of formula II

or a pharmaceutically acceptable equivalent of said compound, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen or C₁-C₃alkyl; and

A¹, A², A³, A⁴ and A⁵ are independently hydrogen, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₃ perhaloalkyl, phenyl, phenoxy, benzyl, benzyloxy, hydroxy,halo, cyano, nitro, —SO₂R⁹, —(C═O)NR⁹R¹⁰, —(C═O)NR⁹(CH₂)_(n)COOH,—NR⁹(C═O)R¹⁰, —(CH₂)_(n)COOH or —COOH, or any adjacent two of A¹, A²,A³, A⁴ and A⁵ form with the benzene ring a fused 5- or 6-memberedcarbocyclic or heterocyclic aromatic ring, said heterocyclic aromaticring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s)

R⁹ and R¹⁰ are independently hydrogen, C₁-C₆ alkyl, phenyl or benzyl;and

n is 1-3;

provided that if A¹, A³ and A⁵ are independently hydrogen, C₁-C₆ alkyl,C₁-C₆ alkoxy, halo, nitro, phenyl, phenoxy, benzyl, benzyloxy or —COOH,then neither A² nor A⁴ are —COOH; and provided that if any adjacent twoof A³, A⁴ and A⁵ form with the benzene ring a fused 5- or 6-memberedcarbocyclic or heterocyclic aromatic ring, said heterocyclic aromaticring containing 1 or 2 oxygen, nitrogen and/or sulfur heteroatom(s),then A² is not —COOH.

In one embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are each hydrogen;A¹, A², A³, A⁴ and A⁵ are independently hydrogen, C₁-C₄ alkyl, C₁-C₂alkoxy, C₁-C₂ perhaloalkyl, phenyl, phenoxy, hydroxy, halo, cyano,nitro, —SO₂R⁹, —(C═O)NR⁹R¹⁰, —(C═O)NR⁹(CH₂)COOH, —NR⁹(C═O)R¹⁰ or—(CH₂)COOH; and R⁹ and R¹⁰ are independently hydrogen, methyl or benzyl.

In another embodiment, any adjacent two of A¹, A², A³, A⁴ and A⁵ formwith the benzene ring a fused 5- or 6-membered carbocyclic orheterocyclic aromatic ring, said heterocyclic aromatic ring containing 1or 2 oxygen, nitrogen and/or sulfur heteroatom(s).

This invention also relates to a compound of formula III

or a pharmaceutically acceptable equivalent of said compound, wherein:

X and Y are independently —CR⁵R⁶—, —O—, —S— or —NR—, provided that atleast one of X and Y is/are —CR⁵R⁶—;

A¹, A², A³, A⁴ and A⁵ are independently hydrogen, C₁-C₉ alkyl, C₂-C₉alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle, heterocycle, C₁-C₉alkoxy, C₂-C₉ alkenyloxy, phenoxy, benzyloxy, hydroxy, halo, nitro,cyano, isocyano, —COOR⁷, —COR⁷, —NR⁷R⁸, —SR⁷, —SOR⁷, —SO₂R⁷, —SO₂(OR⁷)—(C═O)NR⁷R⁸, —(C═O)NR⁷(CH₂)_(n)COOH, —NR⁷(C═O)R⁸ or —(CH₂)_(n)COOH, orany adjacent two of A¹, A², A³, A⁴ and A⁵ form with the benzene ring afused ring that is saturated or unsaturated, aromatic or non-aromatic,and carbocyclic or heterocyclic, said heterocyclic ring containing 1 or2 oxygen, nitrogen and/or sulfur heteroatom(s);

n is 1-3;

R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, C₁-C₉alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle orheterocycle; and

said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle,alkoxy, alkenyloxy, phenoxy, benzyloxy, and fused ring are independentlyunsubstituted or substituted with one or more substituent(s);

provided that if A¹, A² and A³ are each hydrogen, and A⁴ and A⁵ are each—COOH, then A⁴ is ortho to A⁵; and provided that if Y is —CR⁵R⁶—, thenat least one of A¹, A², A³, A⁴ and A⁵ is/are independently phenoxy,benzyloxy, aryl, heteroaryl, carbocycle or heterocycle that issubstituted with one or more substituent(s).

In one embodiment, Y is —O—, —S— or —NR—; A¹, A², A³, A⁴ and A⁵ areindependently hydrogen, C₁-C₄ alkyl, C₁-C₂ alkoxy, hydroxy, halo, —COOH,—COR⁷, —NR⁷(C═O)R⁸ or —(CH₂)COOH; and R⁷ and R⁸ are independentlyhydrogen or methyl.

In another embodiment, Y is —CR⁵R⁶—; A¹, A², A³ and A⁴ are eachhydrogen; and A⁵ is phenoxy, benzyloxy, aryl, heteroaryl, carbocycle orheterocycle, wherein said phenoxy and benzyloxy are substituted with—COOH, and said aryl, heteroaryl, carbocycle and heterocycle aresubstituted with one or more substituent(s) selected from the groupconsisting of cyano and —COOH.

Possible substituents of said alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocycle, heterocycle, alkoxy, alkenyloxy, phenoxy, benzyloxy, andfused ring include, without limitation, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy,hydroxy, carboxy, hydroperoxy, carbamido, carbamoyl, carbamyl, carbonyl,carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano,cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano,nitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino,oxo, C₁-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl,sulfinyl, sulfo, sulfonyl, thiocarboxy, thiocyano, isothiocyano,thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl,trifluoromethyl, iodosyl, iodyl, phosphino, phosphinyl, phospho,phosphono, arsino, selanyl, disilanyl, siloxy, silyl, silylene andcarbocyclic and heterocyclic moieties. Carbocyclic moieties includealicyclic and aromatic structures.

Examples of carbocyclic and heterocyclic moieties include, withoutlimitation, phenyl, benzyl, naphthyl, indenyl, azulenyl, fluorenyl,anthracenyl, indolyl, isoindolyl, indolinyl, benzofuranyl,benzothiophenyl, indazolyl, benzimidazolyl, benzthiazolyl,tetrahydrofuranyl, tetrahydropyranyl, pyridyl, pyrrolyl, pyrrolidinyl,pyridinyl, pyrimidinyl, purinyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, quinolizinyl, furyl, thiophenyl, imidazolyl,oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isotriazolyl,oxadiazolyl, triazolyl, thiadiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, trithianyl, indolizinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, thienyl, tetrahydroisoquinolinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl.

Representative compounds of the invention are set forth below in TABLEI.

TABLE I Compound No. Structure/Name 1

alpha-(3-mercaptopropyl)-3-(trifluoromethyl)- benzenepropanoic acid 2

alpha-(3-mercaptopropyl)-benzenepropanoic acid 3

4-hydroxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 4

2,3,4,5,6-pentafluoro-alpha-(3-mercaptopropyl)- benzenepropanoic acid 5

3-carboxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 6

4-carboxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 7

alpha-(3-mercaptopropyl)-4-(methylsulfonyl)- benzenepropanoic acid 8

2-cyano-alpha-(3-mercaptopropyl)- benzenepropanoic acid 9

5-(2-carboxy-5-mercaptopentyl)-1,3- benzenedicarboxylic acid 10

5-carboxy-2-chloro-alpha-(3-mercaptopropyl)- benzenepropanoic acid 11

3-carboxy-4-fluoro-alpha-(3-mercaptopropyl)- benzenepropanoic acid 12

4-(2-cyanophenyl)-alpha-(3-mercaptopropyl)- benzenepropanoic acid 13

2-(aminocarbonyl)-alpha-(3-mercaptopropyl)- benzenepropanoic acid 14

3-(1-carboxy-4-mercaptobutoxy)-benzoic acid 15

5-mercapto-2-phenoxy-pentanoic acid 16

2-(3,5-dimethoxyphenoxy)-5-mercapto-pentanoic acid 17

alpha-(3-mercaptopropyl)-2,5-dimethoxy- benzenepropanoic acid 18

alpha-(3-mercaptopropyl)-3-phenoxy- benzenepropanoic acid 19

2-(3-hydroxyphenoxy)-5-mercapto-pentanoic acid 20

3-(1-carboxy-4-mercaptobutoxy)-benzeneacetic acid 21

4-(1-carboxy-4-mercaptobutoxy)-benzeneacetic acid 22

alpha-(3-mercaptopropyl)-4-phenyl- benzenepropanoic acid 23

2-(3-acetylphenoxy)-5-mercapto-pentanoic acid 24

2-[3-(acetylamino)phenoxy]-5-mercapto-pentanoic acid 25

2-(4-acetylphenoxy)-5-mercaptopentanoic acid 26

4-(acetylamino)-alpha-(3-mercaptopropyl)- benzenepropanoic acid 27

3-(1-carboxy-4-mercaptobutoxy)-4-methoxy-benzoic acid 28

4-(carboxymethyl)-alpha-(3-mercaptopropyl)- benzenepropanoic acid 29

2-(1-carboxy-4-mercaptobutoxy)-benzoic acid 30

4-(1-carboxy-4-mercaptobutoxy)-benzoic acid 31

3-carboxy-2-chloro-alpha-(3-mercaptopropyl)- benzenepropanoic acid 32

3-carboxy-4-chloro-alpha-(3-mercaptopropyl)- benzenepropanoic acid 33

3-(1-carboxy-4-mercaptobutoxy)-4-chloro-benzoic acid 34

3-(1-carboxy-4-mercaptobutoxy)-4-fluoro-benzoic acid 35

5-carboxy-2-fluoro-alpha-(3-mercaptopropyl)- benzenepropanoic acid 36

5-carboxy-alpha-(3-mercaptopropyl)-2-methoxy- benzenepropanoic acid 37

4-carboxy-alpha-(3-mercaptopropyl)-1- naphthalenepropanoic acid 38

2-carboxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 39

4-carboxy-2,3,5,6-tetrafluoro-alpha-(3- mercaptopropyl)-benzenepropanoicacid 40

5-mercapto-2-(phenylthio)-pentanoic acid 41

3-[1-carboxy-4-mercaptobutyl)thio]-benzoic acid 42

alpha-(3-mercaptopropyl)-2-naphthalenepropanoic acid 43

2-chloro-alpha-(3-mercaptopropyl)- benzenepropanoic acid 44

alpha-(3-mercaptopropyl)-3-[[(phenylmethyl)amino]carbonyl]-benzenepropanoic acid 45

3-bromo-5-carboxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 46

3-[[(carboxymethyl)amino]carbonyl]-alpha-(3-mercaptopropyl)-benzenepropanoic acid 47

3-bromo-4-carboxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 48

3-carboxy-alpha-(3-mercaptopropyl)-5-nitro- benzenepropanoic acid 49

3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)-benzenepropanoic acid 50

5-carboxy-alpha-(3-mercaptopropyl)-2-nitro- benzenepropanoic acid 51

3′-(2-carboxy-5-mercaptopentyl)-[1,1′-biphenyl]- 3-carboxylic acid 52

2-bromo-5-carboxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 53

(+)-3-carboxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 54

(−)-3-carboxy-alpha-(3-mercaptopropyl)- benzenepropanoic acid 55

5-(2-carboxy-5-mercaptopentyl)-[1,1′-biphenyl]- 3-carboxylic acid 56

2-(2-carboxy-5-mercaptopentyl)-[1,1′-biphenyl]- 4-carboxylic acid 57

6-(2-carboxy-5-mercaptopentyl)-[1,1′-biphenyl]- 2-carboxylic acid 58

4-(2-carboxy-5-mercaptopentyl)-[1,1′-biphenyl]- 2-carboxylic acid 59

3-carboxy-alpha-(3-mercaptopropyl)-5-methoxy- benzenepropanoic acid 60

3′-(2-carboxy-5-mercaptopentyl)-[1,1′-biphenyl]- 2-carboxylic acid 61

3-(2-carboxyphenoxy)-alpha-(3-mercaptopropyl)- benzenepropanoic acid 62

4-(2-carboxyphenoxy)-alpha-(3-mercaptopropyl)- benzenepropanoic acid 63

3-carboxy-alpha-(3-mercaptobutyl)- benzenepropanoic acid 64

4′-(2-carboxy-5-mercaptopropyl)-[1,1′-biphenyl]- 2-carboxylic acid 65

3-carboxy-alpha-(3-mercaptopropyl)-5- (phenylmethoxy)-benzenepropanoicacid 66

alpha-(3-mercaptopropyl)-3-phenyl- benzenepropanoic acid 67

3-carboxy-alpha-(3-mercaptopropyl)-5-phenoxy- benzenepropanoic acid 68

3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptobutyl)-benzenepropanoic acid 69

3-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)- benzenepropanoic acid 70

3-(1-carboxy-4-mercaptobutoxy)-5-(1,1- dimethylethyl)-benzoic acid 71

3-[(1-carboxy-4-mercaptobutyl)thio-5-(1,1- dimethylethyl)-benzoic acid72

3-[(1-carboxy-4-mercaptobutyl)amino]-5-(1,1- dimethylethyl)-benzoic acid

The inventive compounds possess one or more asymmetric carbon center(s)and are thus capable of existing in the form of optical isomers as wellas in the form of racemic or non-racemic mixtures of optical isomers.The optical isomers can be obtained by resolution of the racemicmixtures according to conventional processes well known in the art, forexample by formation of diastereoisomeric salts by treatment withanoptically active acid or base and then separation of the mixture ofdiastereoisomers by crystallization followed by liberation of theoptically active bases from these salts. Examples of useful acidsinclude tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaricand camphorsulfonic acids.

A different process for separating optical isomers involves the use of achiral chromatography column optimally chosen to maximize the separationof the enantiomers. Still another available method involves synthesis ofcovalent diastereoisomeric molecules, for example, esters, amides,acetals, ketals, and the like, by reacting compounds used in theinventive methods and pharmaceutical compositions with an opticallyactive acid in an activated form, an optically active diol or anoptically active isocyanate. The synthesized diastereoisomers can beseparated by conventional means, such as chromatography, distillation,crystallization or sublimation, and then hydrolyzed to deliver theenantiomerically pure compound. In some cases, hydrolysis to the parentoptically active drug prior to dosing the patient is unnecessary sincethe compound can behave as a prodrug. The optically active compounds ofthe invention can likewise be obtained by utilizing optically activestarting materials.

It is understood that the inventive compounds encompass optical isomersas well as racemic and non-racemic mixtures.

METHODS OF THE INVENTION Methods for Inhibiting NAALADase EnzymeActivity

This invention relates to a method for inhibiting NAALADase enzymeactivity in an animal or a mammal, comprising administering to saidanimal or mammal an effective amount of a compound of the invention, asdefined above.

Methods for Treating Glutamate Abnormalities

This invention further relates to a method for treating a glutamateabnormality in an animal or a mammal, comprising administering to saidanimal or mammal an effective amount of a compound of the invention, asdefined above.

Glutamate abnormalities to be treated may be selected from the groupconsisting of compulsive disorder, stroke, ischemia, demyelinatingdisease, Parkinson's disease, ALS, Huntington's disease, schizophrenia,pain, anxiety, anxiety disorder, memory impairment and glaucoma.Preferably, the compulsive disorder is alcohol, nicotine or cocainedependence.

Stroke patients often experience a significant temporal delay betweenthe onset of ischemia and the initiation of therapy. Thus, there is aneed for neuroprotectants with a long therapeutic window of opportunity.It is expected that the inventive compounds have a therapeutic window ofopportunity of at least 1 hour. Accordingly, when the glutamateabnormality is stroke, the compound of the invention may be administeredto said animal or mammal for up to 60 minutes, 120 minutes or morefollowing onset of stroke.

Without being bound to any particular mechanism of action, preferredcompounds of the invention are expected to be those that block glutamaterelease pre-synaptically without interacting with post-synapticglutamate receptors. Such compounds would be devoid of the behavioraltoxicities associated with post-synaptic glutamate antagonists.

Methods for Effecting Neuronal Activities

This invention further relates to a method for effecting a neuronalactivity in an animal or a mammal, comprising administering to saidanimal or mammal an effective amount of a compound of the invention, asdefined above.

The neuronal activity that is effected by the inventive method may bestimulation of damaged neurons, promotion of neuronal regeneration,prevention of neurodegeneration or treatment of a neurological disorder.

Examples of neurological disorders that are treatable by the inventivemethods include without limitation: trigeminal neuralgia;glossopharyngeal neuralgia; Bell's Palsy; myasthenia gravis; musculardystrophy; ALS; progressive muscular atrophy; progressive bulbarinherited muscular atrophy; herniated, ruptured or prolapsedinvertebrate disk syndromes; cervical spondylosis; plexus disorders;thoracic outlet destruction syndromes; peripheral neuropathies such asthose caused by lead, dapsone, ticks, porphyria, or Guillain-Barrésyndrome; diabetic neuropathy; pain; Alzheimer's disease; andParkinson's disease.

The inventive method is particularly useful for treating a neurologicaldisorder selected from the group consisting of peripheral neuropathycaused by physical injury or disease state, diabetic neuropathy, pain,traumatic brain injury, physical damage to spinal cord, strokeassociated with brain damage, demyelinating disease and neurologicaldisorder relating to neurodegeneration.

When the neurological disorder is pain, the compound of the invention ispreferably administered in combination with an effective amount ofmorphine.

Examples of neurological disorders relating to neurodegeneration includeAlzheimer's disease, Parkinson's disease, and ALS.

Methods for Treating Prostate Diseases

This invention further relates to a method for treating a prostatedisease in an animal or a mammal, comprising administering to saidanimal or mammal an effective amount of a compound of the invention, asdefined above.

Methods for Treating Cancers

This invention further relates to a method for treating cancer in ananimal or a mammal, comprising administering to said animal or mammal aneffective amount of a compound of the invention, as defined above.

Preferred cancers to be treated are those in tissues where NAALADaseresides, including without limitation the brain, kidney and testis.

Methods for Inhibiting Angiogenesis

This invention further relates to a method for inhibiting angiogenesisin an animal or a mammal, comprising administering to said animal ormammal an effective amount of a compound of the invention, as definedabove.

Angiogenesis may be necessary for fertility or metastasis of cancertumors, or may be related to an angiogenic-dependent disease. Thus, theinventive methods may also be useful for treating anangiogenic-dependent disease including, without limitation, rheumatoidarthritis, cardiovascular diseases, neovascular diseases of the eye,peripheral vascular disorders, dermatologic ulcers and cancerous tumorgrowth, invasion or metastasis.

Methods for Effecting TGF-β Activity

This invention further relates to a method for effecting a TGF-βactivity in an animal or a mammal, comprising administering to saidanimal or mammal an effective amount of a compound of the invention, asdefined above.

Said effecting a TGF-β activity includes increasing, reducing orregulating TGF-β levels, and treating TGF-β abnormalities. Examples ofTGF-β abnormalities to be treated include neurodegenerative disorders,extra-cellular matrix formation disorders, cell-growth related diseases,infectious diseases, immune related diseases, epithelial tissuescarring, collagen vascular diseases, fibroproliferative disorders,connective tissue disorders, inflammation, inflammatory diseases,respiratory distress syndrome, infertility and diabetes.

Typical neurodegenerative disorders to be treated include neural tissuedamage resulting from ischemia reperfusion injury, myelination andneurodegeneration.

Typical cell-growth related disorders to be treated include thoseaffecting kidney cells, hematopoietic cells, lymphocytes, epithelialcells and endothelial cells.

Typical infectious diseases to be treated include those caused by amacrophage pathogen, particularly a macrophage pathogen selected fromthe group consisting of bacteria, yeast, fungi, viruses, protozoa,Trypanosoma cruzi, Histoplasma capsulatum, Candida albicans, Candidaparapsilosis, Cryptococcus neoformans, Salmonella, Pneumocystis,Toxoplasma, Listeria, Mycobacteria, Rickettsia and Leishmania.Mycobacteria include without limitation Mycobacterium tuberculosis andMycobacterium leprae. Toxoplasma includes without limitation Toxoplasmagondii. Rickettsia includes without limitation R. prowazekii, R. coroniiand R. tsutsugamushi.

Other examples of infectious diseases to be treated include single ormultiple cutaneous lesions, mucosal disease, Chagas' disease, acquiredimmunodeficiency syndrome (AIDS), toxoplasmosis, leishmaniasis,trypanosomiasis, shistosomiasis, cryptosporidiosis, Mycobacterium aviuminfections, Pneumocystis carinii pneumonia and leprosy.

Typical immune related diseases to be treated include autoimmunedisorders; impaired immune function; and immunosuppression associatedwith an infectious disease, particularly, trypanosomal infection, viralinfection, human immunosuppression virus, human T cell lymphotropicvirus (HTLV-1), lymphocytic choriomeningitis virus or hepatitis.

Typical collagen vascular diseases to be treated include progressivesystemic sclerosis (“PSS”), polymyositis, scleroderma, dermatomyositis,eosinophilic fascitis, morphea, Raynaud's syndrome, interstitialpulmonary fibrosis, scleroderma and systemic lupus erythematosus.

Typical fibroproliferative disorders to be treated include diabeticnephropathy, kidney disease, proliferative vitreoretinopathy, livercirrhosis, biliary fibrosis, and myelofibrosis. Especially preferredkidney diseases include mesangial proliferative glomerulonephritis,crescentic glomerulonephritis, diabetic neuropathy, renal interstitialfibrosis, renal fibrosis in transplant patients receiving cyclosporin,and HIV-associated nephropathy.

Typical connective tissue disorders to be treated include scleroderma,myelofibrosis, and hepatic, intraocular and pulmonary fibrosis.

Typical inflammatory diseases to be treated are associated with PSS,polymyositis, scleroderma, dermatomyositis, eosinophilic fascitis,morphea, Raynaud's syndrome, interstitial pulmonary fibrosis,scleroderma, systemic lupus erythematosus, diabetic nephropathy, kidneydisease, proliferative vitreoretinopathy, liver cirrhosis, biliaryfibrosis, myelofibrosis, mesangial proliferative glomerulonephritis,crescentic glomerulonephritis, diabetic neuropathy, renal interstitialfibrosis, renal fibrosis in transplant patients receiving cyclosporin,or HIV-associated nephropathy.

Without being limited to any particular mechanism of action, preferredcompounds of this invention treat inflammatory diseases by regulatingTGF-β and/or inhibiting myeloperoxidase.

Other uses associated with the inventive compounds' TGF-β regulatingproperties include:

stimulating growth of tissue, glands or organs, particularly growth thatwould enhance milk production or weight gain;

stimulating cell proliferation, particularly proliferation offibroblasts, mesenchymal cells or epithelial cells;

inhibiting cell growth, particularly of epithelial cells, endothelialcells, T and B lymphocytes and thymocytes;

inhibiting expression of adipose, skeletal muscle and hematopoieticphenotypes, neoplasms, non-cytocidal viral or other pathogenicinfections and autoimmune disorders;

mediating disease resistance and susceptibility;

suppressing cellular immune response;

inhibiting scar tissue formation, preferably in skin or other epithelialtissue that has been damaged by wounds resulting from accidental injury,surgical operations, trauma-induced lacerations or other trauma, orwounds involving the peritoneum for which the excessive connectivetissue formation is abdominal adhesions;

increasing the effectiveness of a vaccine, particularly a vaccine for anallergy towards, for example, dust or hayfever; and

inhibiting polyp formation.

Diagnostic Methods and Kits

The inventive compounds are useful for in vitro and in vivo diagnosticmethods for detecting diseases, disorders and conditions where NAALADaselevels are altered including, without limitation, neurologicaldisorders, glutamate abnormalities, diabetic neuropathy, pain,compulsive disorders, prostate diseases, cancers, TGF-β abnormalitiesand glaucoma.

Accordingly, this invention also relates to a method for detecting adisease, disorder or condition where NAALADase levels are altered,comprising:

(i) contacting a sample of bodily tissue or fluid with a compound of theinvention, as defined above, wherein said compound binds to anyNAALADase in said sample; and

(ii) measuring the amount of any NAALADase bound to said sample, whereinthe amount of NAALADase is diagnostic for said disease, disorder orcondition.

Examples of bodily tissues and fluids include, without limitation,prostate tissue, ejaculate, seminal vesicle fluid, prostatic fluid,urine, blood, saliva, tears, sweat, lymph and sputum.

The compound may be labeled with a marker using techniques known in theart. Useful markers include, without limitation, enzymatic markers andimaging reagents. Examples of imaging reagents include radiolabels suchas ¹³¹I, ¹¹¹In, ¹²³I, ⁹⁹Tc, ³²P, ¹²⁵I, ³H and ¹⁴C; fluorescent labelssuch as fluorescein and rhodamine; and chemiluminescers such asluciferin.

The amount of NAALADase can be measured using techniques known in theart including, without limitation, assays (such as immunometric,calorimetric, densitometric, spectrographic and chromatographic assays)and imaging techniques (such as magnetic resonance spectroscopy (“MRS”),magnetic resonance imaging (“MRI”), single-photon emission computedtomography (“SPECT”) and positron emission tomography (“PET”)).

This invention further relates to a diagnostic kit for detecting adisease, disorder or condition where NAALADase levels are altered,comprising a compound of the invention, as defined above, labeled with amarker. The kit may further comprise buffering agents, agents forreducing background interference, control reagents and/or apparatus forconducting the test.

This invention further relates to a method for detecting a disease,disorder or condition where NAALADase levels are altered in an animal ora mammal, comprising:

(i) labeling a compound of the invention, as defined above, with animaging reagent;

(ii) administering to said animal or mammal an effective amount of thelabeled compound;

(iii) allowing said labeled compound to localize and bind to NAALADasepresent in said animal or mammal; and

(iv) measuring the amount of NAALADase bound to said labeled compound,wherein the amount of NAALADase is diagnostic for said disease, disorderor condition.

The amount of NAALADase can be measured in vivo using known imagingtechniques, as described above.

Incorporation by Reference

The relationship between NAALADase inhibitors and glutamate, and theeffectiveness of NAALADase inhibitors in treating and detecting variousdiseases, disorders and conditions have been discussed in U.S. Pat. Nos.5,672,592, 5,795,877, 5,804,602, 5,824,662, 5,863,536, 5,977,090,5,981,209, 6,011,021, 6,017,903, 6,025,344, 6,025,345, 6,046,180,6,228,888 and 6,265,609; International Publications Nos. WO 00/01668 andWO 00/38785; and other references generally known in the art. Thepresent inventors hereby incorporate by reference, as though set forthherein in full, the entire contents of the aforementioned patents,patent applications and publications, particularly their discussions,figures and data regarding the effectiveness of NAALADase inhibitors ininhibiting angiogenesis, in effecting TGF-β activity, in diagnosingdiseases, and in treating ischemia, spinal cord injury, demyelinatingdiseases, Parkinson's disease, ALS, alcohol dependence, nicotinedependence, cocaine dependence, prostate disease, cancer, diabeticneuropathy, pain, schizophrenia, anxiety, anxiety disorder and memoryimpairment. The present inventors have discovered that the inventivecompounds are effective NAALADase inhibitors. Thus, the inventivecompounds are expected to have the same uses as the NAALADase inhibitorsdisclosed in the patents, patent applications and publicationsincorporated by reference.

Pharmaceutical Compositions of the Invention

This invention also relates to a pharmaceutical composition comprising:

(i) an effective amount of a compound of the invention; and

(ii) a pharmaceutically acceptable carrier.

Preferably, the compound of the invention is present in an effectiveamount for inhibiting NAALADase enzyme activity or angiogenesis,effecting a neuronal activity or TGF-β activity, or treating a glutamateabnormality, compulsive disorder, prostate disease, cancer or glaucomain an animal or a mammal.

Route of Administration

The inventive compounds and compositions may be administered locally orsystemically by any means known to an ordinarily skilled artisan. Forexample, the inventive compounds and compositions may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir in dosage formulationscontaining conventional non-toxic pharmaceutically acceptable carriers,adjuvants and vehicles. The term parenteral as used herein includessubcutaneous, intravenous, intraarterial, intramuscular,intraperitoneal, intrathecal, intraventricular, intrasternal,intracranial or intraosseous injection and infusion techniques. Theexact administration protocol will vary depending upon various factorsincluding the age, body weight, general health, sex and diet of thepatient; the determination of specific administration procedures wouldbe routine to an ordinarily skilled artisan.

To be effective therapeutically as central nervous system targets, theinventive compounds and compositions should readily penetrate theblood-brain barrier when peripherally administered. Compounds thatcannot penetrate the blood-brain barrier can be effectively administeredby an intraventricular route or by other methods recognized in the art.See, for example, U.S. Pat. Nos. 5,846,565, 5,651,986 and 5,626,862.

Dosage

The inventive compounds and compositions may be administered by a singledose, multiple discrete doses or continuous infusion. Pump means,particularly subcutaneous pump means, are preferred for continuousinfusion.

Dose levels on the order of about 0.001 to about 10,000 mg/kg of theactive ingredient compound are useful in the treatment of the aboveconditions, with preferred levels being about 0.1 to about 1,000 mg/kg,and more preferred levels being about 1 to about 100 mg/kg. The specificdose level for any particular patient will vary depending upon a varietyof factors, including the activity and the possible toxicity of thespecific compound employed; the age, body weight, general health, sexand diet of the patient; the time of administration; the rate ofexcretion; drug combination; the severity of the particular diseasebeing treated; and the form of administration. Typically, in vitrodosage-effect results provide useful guidance on the proper doses forpatient administration. Studies in animal models are also helpful. Theconsiderations for determining the proper dose levels are well known inthe art.

Administration Regimen

Any administration regimen well known to an ordinarily skilled artisanfor regulating the timing and sequence of drug delivery can be used andrepeated as necessary to effect treatment. Such regimen may includepretreatment and/or co-administration with additional therapeuticagents.

Co-Administration with Other Treatments

The inventive compounds and compositions may be used alone or incombination with one or more additional agent(s) for simultaneous,separate or sequential use.

The additional agent(s) may be any therapeutic agent(s) known to anordinarily skilled artisan, including without limitation: one or morecompound(s) of the invention; steroids, for example, hydrocortisonessuch as methylprednisolone; anti-inflammatory or anti-immune drugs, suchas methotrexate, azathioprine, cyclophosphamide or cyclosporin A;interferon-β; antibodies, such as anti-CD4 antibodies; agents which canreduce the risk of a second ischemic event, such as ticlopidine;chemotherapeutic agents; immunotherapeutic compositions; electromagneticradiosensitizers; and morphine.

The inventive compounds and compositions can be co-administered with oneor more therapeutic agents either together in a single formulation, orseparately in individual formulations designed for optimal release ratesof their respective active agent. Each formulation may contain fromabout 0.01% to about 99.99% by weight, preferably from about 3.5% toabout 60% by weight, of a compound of this invention, as well as one ormore pharmaceutically acceptable carriers.

Preparation of Compounds

The inventive compounds can be readily prepared by standard techniquesof organic chemistry, utilizing the general synthetic pathways depictedbelow in SCHEMES I, II, and III. Precursor compounds are eithercommercially available or may be prepared by methods known to a personof skill in the art.

EXAMPLES

The following examples are illustrative of this invention and are notintended to be limitations thereon. Unless otherwise indicated, allpercentages are based upon 100% by weight of the final composition.

Example 1 Preparation of5-carboxy-2-chloro-alpha-(3-mercaptopropyl)-benzenepropanoic acid 10(Scheme I)

Methyl 3-bromomethyl-4-chlorobenzoate II

To a suspension of methyl 4-chloro-3-methylbenzoate I (19.9 g, 108 mmol)and N-bromosuccinimide (NBS, 20.2 g, 114 mmol) in carbon tetrachloride(500 mL) was added benzoyl peroxide (1.30 g, 5.4 mmol), and the mixturewas stirred at 90° C. overnight. The mixture was then cooled and thewhite precipitate was removed by filtration. The filtrate wasconcentrated and the resulting solid was re-crystallized from ethylacetate to give methyl 3-bromomethy-4-chlorobenzoate II (15.0 g, 57mmol, 53%) as a white solid: ¹H NMR (CDCl₃) δ 3.95 (s, 3H), 4.63 (s,2H), 7.49 (d, J=8.3 Hz, 1H), 7.94 (dd, J=2.1, 8.3 Hz, 1H), 8.15 (d,J=2.1 Hz, 1H).

3-(2-Chloro-5-methoxycarbonylbenzyl)-tetrahydrothiopyrane-2-one IV

To a solution of lithium diisopropylamide (2.0 M solution, 3.3 mL, 6.6mmol) in THF (25 mL) was added tetrahydrothiopyran-2-one III (0.731 g,6.3 mmol) at −40° C., and the mixture was stirred at −40° C. for 45minutes. A solution of methyl 3-bromomethy-4-chlorobenzoate II (1.67 g,6.3 mmol) in THF (10 mL) was then dropwise added to the mixture at −40°C. Subsequently, hexamethylphosphoramide (0.20 g, 1.4 mmol) was added tothe mixture at −40° C., and the reaction mixture was stirred at −40° C.for 4 hours., A saturated ammonium chloride solution (30 mL) was addedto the reaction mixture, and the organic solvent was removed underreduced pressure. The mixture was then partitioned between ether (150mL) and H₂O (150 mL). The organic layer was washed with brine, driedover MgSO₄, and concentrated. The crude material was chromatographed onsilica gel using EtOAc/hexanes to afford3-(2-chloro-5-methoxycarbonylbenzyl)-tetrahydrothio-pyrane-2-one IV(0.60 g, 2.0 mmol, 32%) as a white solid: ¹H NMR (CDCl₃) δ 1.65-1.75 (m,1H), 1.90-2.05 (m, 2H), 2.05-2.15 (m, 2H), 2.74 (dd, J=9.4, 13.9 Hz,1H), 2.85-3.00 (m, 1H), 3.10-3.20 (m, 2H), 3.58 (dd, J=4.7, 13.9 Hz,1H), 3.92 (s, 3H), 7.44 (d, J=8.3 Hz, 1H), 7.85 (dd, J=8.3, 2.1 Hz, 1H),7.91 (d, J=2.1 Hz, 1H).

5-Carboxy-2-chloro-alpha-(3-mercaptopropyl) benzenepropanoic acid 10

A solution of3-(2-Chloro-5-methoxycarbonylbenzyl)-tetrahydrothiopyrane-2-one IV (9.26g, 31.0 mmol) in THF (70 mL) was purged for 15 minutes with nitrogen. Adegassed aqueous sodium hydroxide solution (2.2 M, 70 mL, 154 mmol) wasadded to the solution and the mixture was stirred at room temperatureunder nitrogen overnight. The reaction mixture was washed with ether,acidified by 3N HCl at 0° C., and extracted with ether. The extract wasdried over MgSO₄ and concentrated to afford5-carboxy-2-chloro-alpha-(3-mercaptopropyl) benzenepropanoic acid 10(8.42 g, 27.8 mmol, 90%) as a white solid: ¹H NMR (CD₃OD) δ 1.50-1.80(m, 4H), 2.35-2.50 (m, 2H), 2.65-2.75 (m, 1H), 2.91 (dd, J=6.2, 13.8 Hz,1H), 2.96 (dd, J=8.8, 13.8 Hz, 1 H), 7.39 (d, J=8.3 Hz, 1H), 7.76 (dd,J=2.0, 8.3 Hz, 1H), 7.86 (d, J=2.0 Hz, 1H); ¹³C NMR (CD₃OD) δ 25.1,32.5, 33.2, 37.4, 46.8, 130.8, 131.2, 134.0, 139.1, 140.5, 169.2, 178.9.Anal. Calcd for C₁₃H₁₅ClO₄S: C, 51.57; H, 4.99; S, 10.59; Cl, 11.71.Found: C, 51.59; H, 4.94; S, 10.43; Cl, 11.80.

Example 2 Preparation of3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)-benzenepropanoicacid 49 (Scheme II)

Methyl 5-tert-butylhydrogenisophthalate VI

To a solution of dimethyl 5-tert-butylisophthalate V (23.0 g, 92 mmol)in methanol (150 mL) was added a solution of sodium hydroxide (3.68 9,92 mmol) in H₂O (10 mL) at 25° C., and the mixture was stirred at 25° C.for 3 hours. The organic solvent was removed under reduced pressure andthe residual solid was suspended in an aqueous sulfuric acid solution(1.0 M). The suspension was filtered and the precipitate was washed withH₂O, dried under vacuum, and crystallized from hexanes/ethyl acetate toafford methyl 5-tert-butylhydrogenisophthalate VI (16.3 g, 69.0 mmol,75%) as a white solid: ¹H NMR (CDCl₃) δ 1.45 (s, 9H), 3.9 (s, 3H), 8.5(s, 1H), 8.7 (s, 1H), 8.8 (s, 1H); ¹³C NMR (CDCl₃) δ 31.3 (3C), 35.2,52.5, 128.8, 129.7, 130.7, 131.1, 131.6, 132.0, 166.7, 171.5.

Methyl 3-tert-butyl-5-hydroxymethylbenzoate VII

Borane-dimethyl sulfide complex (7.23 mL, 76.2 mmol) was slowly added toa solution of methyl 5-tert-butylhydrogenisophthalate VI (12.0 g, 50.8mmol) in THF (100 ml) over the period of 20 minutes at room temperature.The mixture was stirred for 1.5 hours at room temperature and thenrefluxed for 1 additional hour. The reaction mixture was then cooled andthe unreacted borane was decomposed with methanol (10 mL). The solventswere removed under reduced pressure and the residue was dissolved inethyl acetate. The organic solution was washed with a saturated NaHCO₃solution, dried over MgSO₄, and purified by a silica gel columnchromatography (hexane/ethyl acetate) to afford methyl3-tert-butyl-5-hydroxymethylbenzoate VII (10.0 g, 45.0 mmol, 90%) as awhite solid: ¹H NMR (CDCl₃) δ 1.45 (s, 9H), 3.9 (s, 3H), 4.7 (s, 2H),7.6 (s, 1H), 7.8 (s, 1H), 8.0 (s, 1H); ¹³C NMR (CDCl₃) δ 31.4 (3C),35.0, 52.3, 65.3, 125.5, 126.1, 128.8, 130.3, 141.0, 152.1, 167.5.

Methyl 3-bromomethyl-5-tert-butylbenzoate VIII

To a solution of methyl 3-tert-butyl-5-hydroxymethylbenzoate VII (9.50g, 42.7 mmol) and carbon tetrabromide (17.25 g, 52.0 mmol) indichloromethane (50 mL) was slowly added triphenylphosphine (13.6 g,52.0 mmol) over the period of 20 minutes, and the mixture was stirred atroom temperature for 25 minutes. The reaction mixture was concentratedunder reduced pressure and the residue was suspended in ethyl acetate.The precipitate was removed by filtration and the filtrate wasconcentrated. The crude material was purified by a silica gelchromatography (hexanes/ethyl acetate, 4:1), and the product wasre-crystallized form ethyl acetate/hexanes to afford methyl3-bromomethyl-5-tert-butylbenzoate VIII (12.0 g, 42.1 mmol, 99%) as awhite solid: ¹H NMR (CDCl₃) δ 1.45 (s, 9H), 3.7 (s, 3H), 4.4 (s, 2H),7.6 (s, 1H), 7.8 (s, 1H), 8.0 (s, 1H); ¹³C NMR (CDCl₃) δ 31.3 (3C),33.2, 36.0, 52.3, 126.9, 127.5, 130.6, 130.7, 137.9, 152.4, 167.0.

5-(3-tert-butyl-5-methoxycarbonyl-benzyl)-2,2-dimethyl-5-[3-[(triphenylmethyl)thio]propyl]-[1,3]dioxane-4,6-dioneX

A solution of methyl 3-bromomethyl-5-tert-butylbenzoate (10.3 g, 36.1mmol),2,2-dimethyl-5-[3-[(triphenylmethyl)-thio]propyl]-[1,3]dioxane-4,6-dioneIX (13.8 g, 30.0 mmol), and benzyltriethylammonium chloride (6.38 g, 30mmol) in acetonitrile (90 mL) was added potassium carbonate (4.35 g, 30mmol) at 25° C., and the reaction mixture was stirred at 60° C.overnight (the synthesis of compound IX was previously described inInternational Publication No. WO 00/01668). The solvent was removedunder reduced pressure and the residue was partitioned between ethylacetate and a 10% aqueous KHSO₄ solution. The organic layer was driedover MgSO₄, concentrated. The crude material was recrystallized fromethyl acetate/hexane mixture to afford5-(3-tert-butyl-5-methoxycarbonyl-benzyl)-2,2-dimethyl-5-[3-[(triphenyl-methyl)thio]propyl]-[1,3]dioxane-4,6-dioneX (14.0 g, 79%) as a white solid: ¹H NMR (CDCl₃) δ 0.7 (s, 3H), 1.3 (s,9H), 1.2-1.3 (m, 2H), 1.5 (s, 3H), 2.0 (m, 2H), 2.2 (m, 2H), 3.3 (s,2H), 3.8 (s, 3H), 7.2-7.4 (m, 16H), 7.6 (s, 1H), 7.8 (s, 1H); ¹³C NMR(CDCl₃) δ 24.8, 29.1, 29.4, 31.2, 31.4, 34.9, 40.3, 43.7, 52.3, 57.3,66.8, 105.8, 126.0, 126.8, 128.0, 128.5, 129.6, 130.5, 132.3, 135.3,144.8, 152.4, 167.1, 168.5.

2-(3-tert-Butyl-5-methoxycarbonyl-benzyl)-2-[3-[(triphenylmethyl)thio]propyl]-malonicAcid XI

To a solution of5-(3-tert-butyl-5-methoxycarbonyl-benzyl)-2,2-dimethyl-5-[3-[(triphenylmethyl)thio]propyl]-[1,3]dioxane-4,6-dioneX (11 g, 16.5 mmol) in 1,4-dioxane (15 ml) was added a solution ofsodium hydroxide (4.63 g, 115.5 mmol) in H₂O (15 mL) at 25° C., and themixture was stirred at 100° C. for 1 hour. The solvent was removed underreduced pressure and the residue was partitioned between ethyl acetateand a 10% aqueous KHSO₄ solution. The organic layer was dried overMgSO₄, concentrated. The crude material was recrystallized from ethylacetate/hexane mixture to afford2-(3-tert-butyl-5-methoxycarbonyl-benzyl)-2-[3-[(triphenyl-methyl)thio]propyl]malonicacid XI (9.0 g, 90%) as a white solid: ¹H NMR (CD₃OD) δ 1.4 (s, 9H), 1.4(m, 2H), 1.6 (m, 2H), 2.1 (t, J=8.0 Hz, 2H), 3.2 (s, 2H), 7.1-7.4 (m,16H), 7.7 (s, 1H), 7.9 (s, 1H); ¹³C NMR (CD₃OD) δ 24.8, 31.8 (3C), 32.4,33.3, 35.6, 39.0, 59.5, 67.7, 126.2, 127.7, 128.9, 129.6, 130.7, 131.5,132.9, 137.8, 146.2, 152.6, 170.1, 174.5.

2-(3-tert-Butyl-5-methoxycarbonyl-benzyl)-5-[(triphenylmethyl)thio]pentanoicacid XII

A solution of2-(3-tert-butyl-5-methoxycarbonyl-benzyl)-2-[3-[(triphenylmethyl)thio]propyl]-malonicacid XI (6.71 g, 11 mmol) in DMSO (10 ml) was stirred at 130° C. for 1.5hours. The solvent was removed under reduced pressure and water wasadded to the residual oil. The precipitate was filtered off, washed withwater, and dried under vacuum to afford2-(3-tert-butyl-5-methoxycarbonyl-benzyl)-5-[(triphenylmethyl)thio]-pentanoicacid XII (5.86 g, 10.3 mmol, 94%) as a white solid: ¹H NMR (CD₃OD) δ 1.3(s, 9H), 1.3-1.5 (m, 4H), 2.1 (m, 2H), 2.4 (m, 1H), 2.7 (m, 1H), 2.8 (m,1H), 7.1-7.4 (m, 16H), 7.7 (s, 1H), 7.9 (s, 1H); ¹³C NMR (CD₃OD) δ 27.4,31.7 (3C), 32.3, 32.7, 35.6, 39.2, 48.4, 67.7, 125.7, 127.7, 128.6,128.9, 130.8, 131.6, 132.0, 140.8, 146.3, 152.7, 170.3, 178.8.

3-Carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)-benzenepropanoicacid 49

To a solution of2-(3-tert-butyl-5-methoxycarbonyl-benzyl)-5-[(triphenylmethyl)thio]pentanoicacid XII (5.5 g, 9.7 mmol) in dichloromethane (30 mL) were addedtriisopropylsilane (2.4 mL, 11.6 mmol) and trifluoroacetic acid (10 mL),and the mixture was stirred at room temperature for 10 minutes. Thesolvent was removed under reduced pressure and the crude material waspurified by silica gel chromatography (1% AcOH in Hexanes/EtOAc, 4:1) toafford3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)-benzenepropanoicacid 49 (1.7 g, 5.3 mmol, 55%) as a white solid: ¹H NMR (CD₃OD) δ 1.3(s, 9H), 1.5-1.8 (m, 4H), 2.4 (m, 2H), 2.6-2.7 (m, 1H), 2.8-2.9 (m, 1H),2.9-3.0 (m, 1H), 7.5 (s, 1H), 7.7 (s, 1H), 7.8 (s, 1H); ¹³C NMR (CD₃OD)δ 24.8, 31.7 (3C), 31.9, 32.9, 35.6, 39.5, 48.6, 125.7, 128.5, 131.6,132.0, 140.9, 152.8, 170.3, 179.0. Anal. Calcd for C₁₇H₂₄O₄S: C, 62.93;H, 7.46; S, 9.88. Found: C, 63.02; H, 7.36; S, 9.82.

Example 3 Preparation of 3-(1-Carboxy-4-mercaptobutoxy)-benzoic acid 14(Scheme III)

Methyl 3-(4-acetylthio-1-methoxycarbonyl-butoxy)-benzoate XV

To a solution of methyl 2,5-dibromopentanoate XIII (22.00 g, 80.3 mmol)and methyl 3-hydroxybenzoate XIV (10.18 g, 66.9 mmol) in DMF (80 mL) wasadded K₂CO₃ (12.94 g, 93.7 mmol) at room temperature. The mixture wasstirred at room temperature under N₂ for 12 hours, then heated at 70° C.for 1 h. Potassium thioacetate (22.93 g, 200.8 mmol) was added to themixture, which was heated at 70° C. for 2 hours. The mixture -wasallowed to cool to room temperature and was diluted with EtOAc (1000mL). The mixture was washed with H₂O (3×300 mL) and brine (2×300 mL).The organic layer was dried over MgSO₄, filtered and concentrated. Thecrude product was purified by flash chromatography (gradient elution:10% to 20% EtOAc/hexanes) to afford methyl3-(4-acetylthio-1-methoxycarbonyl-butoxy)-benzoate XV (8.40 g, 24.7mmol, 37%) as a yellow oil: R_(f) 0.26 (hexanes/EtOAc, 4:1): ¹H NMR(CDCl₃) δ 1.73-1.88 (m, 2H), 2.01-2.08 (m, 2H), 2.32 (s, 3H), 2.93 (t,J=7.2 Hz, 2H), 3.75 (s, 3H), 3.89 (s, 3H), 4.69 (t, J=6.2 Hz, 1H), 7.07(dm, J=8.0 Hz, 1H), 7.33 (t, J=7.9 Hz, 1H), 7.50 (m, 1H), 7.65 (dm,J=7.7 Hz, 1H); ¹³C NMR (CDCl₃) δ 25.3, 28.3, 30.5, 31.4, 52.1, 52.2,75.8, 115.4, 120.0, 122.8, 129.5, 131.4, 157.5, 166.4, 171.3, 195.4.

3-(1-Carboxy-4-mercaptobutoxy)-benzoic acid 14

A solution of methyl 3-(4-acetylthio-1-methoxy-carbonylbutoxy)benzoateXV (8.00 g, 23.5 mmol) in THF (60 mL) was deoxygenated by bubbling N₂through the solution for 1 hour. To the solution was added deoxygenated3 N NaOH (47 mL, 141 mmol) and the mixture was stirred for 24 hours atroom temperature under N₂. The mixture was acidified with 1 N HCl andextracted with EtOAc (3×300 mL). The organic extracts were washed withwater (300 mL) and brine (300 mL), dried over MgSO₄, filtered, andconcentrated. The residual oil was dissolved in ether and the solutionwas concentrated to afford 3-(1-carboxy-4-mercaptobutoxy)-benzoic acid14 (4.40 g, 16.3 mmol, 69%) as a white solid: ¹H NMR (CDCl₃) δ 1.40 (t,J=7.9 Hz, 1H), 1.83-1.99 (m, 2H), 2.11-2.22 (m, 2H), 2.63 (m, 2H), 4.76(dd, J=7.3, 5.0 Hz, 1H), 7.23 (m, 1H), 7.38 (t, J=7.8 Hz, 1H), 7.49 (m,1H), 7.72 (d, J=7.7 Hz, 1H); ¹³C NMR (CDCl₃) δ 24.1, 29.6, 31.2, 75.5,114.8, 122.1, 123.9, 129.9, 130.5, 157.6, 171.8, 177.1. Anal. Calcd forC₁₂H₁₄O₅S: C, 53.32; H, 5.22; S, 11.86. Found: C, 53.04; H, 5.38; S,11.58.

Example 4 Preparation of(+)-3-carboxy-α-(3-mercaptopropyl)-benzenepropanoic acid 53 and(−)-3-carboxy-α-(3-mercaptopropyl)-benzenepropanoic acid 54

A solution of racemic 3-carboxy-α-(3-mercaptopropyl)-benzenepropanoicacid (2.70 g, 10.1 mmol) was divided into multiple samples of equalvolume, and each of them was passed through a CHIRAPAK AD column (250mm×21 mm id) using carbon dioxide/methanol (77/23, v/v) as eluent at aflow rate of 25 mL/min, at 25° C. The first eluting peak (detected by UVat 290 nm) from each run was combined and concentrated to afford(+)-3-carboxy-α(3-mercaptopropyl)-benzenepropanoic acid 53 (1.02 g, 38%)as a colorless oil: [α]²⁵D=+15.5 (c=1.1, CH3CN).(−)-3-carboxy-α-(3-mercaptopropyl)-benzenepropanoic acid 54 (1.08 g.40%) was obtained likewise from the second peaks as a colorless oil:[α]²⁵D=−13.4 (c=1.1, CH₃CN)

Example 5 In Vitro Inhibition of NAALADase Activity

Various compounds of this invention were tested for in vitro inhibitionof NAALADase activity, and the results are provided below in TABLE II.

TABLE II In Vitro Inhibition of NAALADase Activity Compound No. IC₅₀ 14000 2 2270 3 2140 4 1000 5 17 6 59 7 211 8 450 9 2.55 10 2.09 11 12 12316 13 3950 14 16.3 15 555 16 16,100 17 566 18 308 19 846 20 64 21 82 22229 23 2900 24 27700 25 29.8 26 2200 27 287 28 809 29 4600 30 192 311200 32 175 33 118 34 400 35 6.33 36 26 37 316 38 1700 39 660 40 297 4124 42 1130 43 1350 44 1130 45 2.5 46 286 47 146 48 3 49 0.05 50 8.25 511.83 52 3.33 53 7 54 33.3 56 19 57 70 58 18 59 13 60 184 61 49 62 359063 30 64 87 66 181 67 5 68 1

Protocol for Assaying In Vitro Inhibition of NAALADase Activity

The following were combined in assay tubes: 100 μL of 10 mM COCl₂, 250μL of 200 mM Tris chloride, 100 μL tissue, 100 μL of 10 mM NAALADaseinhibitor in Bakers H₂O, and Bakers H₂O to make a total volume of 950μL. Each assay tube was then incubated for 10 minutes in a 37EC waterbath. 50 μL of 3-H-NAAG was then added to each assay tube and incubatedfor an additional 15 minutes in a 37EC water bath. The assay was stoppedby adding 1.0 ml of 0.1 M sodium phosphate.

Glutamate released by the action of the NAALADase enzyme was separatedfrom the assay solution using an anion exchange resin. The resin wasequilibrated to 25EC, 2.0 ml of the resin was added to a Pasteur pipettepre-loaded with a single glass bead, and each column was washed twicewith distilled H₂O. A column was placed over a scintillation vial and200 μL of an assay sample was loaded onto the column. After draining,glutamate was eluted using two 1.0 ml washes of 1 M formic acid. Afteraddition of 10 ml of scintillation cocktail, each sample was counted for2 minutes on a scintillation counter.

Example 6 In Vitro Assay on Ischemia

To examine the in vitro effect of the inventive compounds on ischemia,cortical cell cultures were treated with various compounds of thisinvention during an ischemic insult utilizing potassium cyanide and2-deoxyglucose, and for one hour thereafter. For a description of theexperimental method used, see Vornov et al., J. Neurochem., Vol. 65, No.4, pp. 1681-1691 (1995). The results are provided below in TABLE III.Neuroprotective effect is expressed as EC₅₀, the concentration of thecompound, which is required to cause a 50% reduction in glutamatetoxicity following an ischemic insult.

TABLE III Compound No. EC₅₀ 5 6.5 6 17.8 7 1060 8 133 9 104 10 7.7 1115.4 12 188 13 10,000 14 16 15 1,000 16 267 17 1400 18 230 20 39.3 2111.6 23 21 26 1800 27 155 28 48 30 399 31 31 32 7 33 110 34 346 35 23 3674.9 37 215 38 762 39 290 40 452 41 409 42 265 43 452 45 2 46 544 47 11948 2.4 50 26 55 1 56 11 57 21 58 16

Example 7 Effect of NAALADase Inhibition on TGF-β in In Vitro IschemiaModel

A NAALAAADase inhibitor, Compound C, was added to ischemia cell culturesto determine its effect on TGF-β levels during stroke. The experimentaldata, set forth in FIGS. 1 and 2, show increased concentrations ofTGF-β1 (FIG. 1) and TGF-β2 (FIG. 2) in ischemic cell cultures treatedwith Compound C. The results indicate that NAALADase inhibition promotesthe release of endogenous TGF-β's from glial cells, which in turnprovides neuroprotection for neighboring neurons.

TGF-β neutralizing antibodies were then added to the ischemic cellcultures. FIG. 3 shows that the TGF-β neutralizing antibodies blockedthe neuroprotective effect of Compound C in the in vitro ischemia model.By contrast, FIG. 4 shows that the addition of another growth factorantibody, FGF antibody, did not block the neuroprotective effect ofCompound C. The results indicate that NAALADase inhibition specificallyaffects TGF-β levels during stroke.

Example 8 Effect of NAALADase Inhibition on TGF-β in In Vivo IschemiaModel

The effect of TGF-β neutralizing antibodies on the neuroprotectiveeffect of Compound C was also studied in rats following MCAO. FIG. 6shows that treatment of MCAO rats with Compound C caused a significantrise in TGF-β1 levels during both occlusion and reperfusion, as assessedby microdialysis. The results indicate that NAALADase inhibitionprovides neuroprotection, at least in part, by regulating endogenousTGF-β's.

Additionally, FIG. 5 shows that TGF-β neutralizing antibodiessignificantly attenuated the neuroprotective effect of Compound C invivo. One of ordinary skill in the art can appreciate that theregulation of TGF-β's by NAALADase inhibitors may have implications notonly in stroke, but also in other diseases, disorders and conditionsincluding, without limitation, neurological diseases, psychiatricdiseases, demyelinating diseases, prostate cancer, inflammation,diabetes and angiogenesis.

Example 9 In Vivo Assay of NAALADase Inhibitors on Neuropathic Pain inSTZ Model

Male Sprague-Dawley rats (200-225 g) were rendered diabetic byintravenous administration of streptozotocin (“STZ”, 70 mg/kg inphosphate buffered saline). Diabetic animals were divided into fivegroups: one group receiving Compound A (10 mg/kg or 1 mg/kg), Compound D(10 mg/kg or 1 mg/kg) or vehicle. Another group of animals(non-STZ-treated) served as non-diabetic controls. Drug/vehicletreatment was started in diabetic animals 45 days post-STZadministration. STZ-induced diabetic rats were tested for sensitivity toa heat source as soon as blood glucose levels rose to 320 mg/dl or above(30 days post STZ). The rats were then acclimated to a Hargreavesapparatus and thermal nociception was monitored using an infrared heatsource directed into the dorsal surface of the hindpaw, and the timetaken for the animal to remove its paw noted to the nearest 0.1 seconds(see Hargreaves et al., supra, for detailed experimental method). Theintensity of the beam source was adjusted such that the mean latency forcontrol animals (non-STZ treated) was approximately 10 seconds. Eachanimal was tested 8 times and the mean difference score (between meannon-diabetic control latency and mean diabetic latency) are graphicallypresented in FIGS. 7A and 7B. Diabetic rats displayed a hyperalgesia(shorter response latency) compared to non-diabetic controls, starting30 days post STZ treatment and progressively worsening in vehicletreated rats. This hyperalgesic response was completely reversed indiabetic rats receiving treatment with Compound D or A (10 mg/kg i.p.daily). Thus, the results show that NAALADase inhibition attenuatesneuropathic pain.

Example 10 In Vivo Assay of NAALADase Inhibitors on Neuropathic Pain inCCI Model

Sciatic nerve ligation, consisting of 4 ligatures tied loosely aroundthe sciatic nerve at 1 mm intervals proximal to the nerve trifurcation,was performed on rats. Following sciatic nerve ligation, the ratsexhibited a thermal hyperalgesia and allodynia. The rats were habituatedto a Hargreaves apparatus. An infrared heat source was directed onto thedorsal surface of each rat's hindpaws and the time taken for the rat towithdraw its paws was noted. The difference in scores between thelatency of the response for the paw on the operated side versus the pawon the unoperated control side was determined.

Compound 49

The rats were treated with either Compound 49 (10, 1 or 0.1 mg/kg) or avehicle for 15 days after sciatic nerve ligation. Thermal pain responseswere measured at days 0, 1, 5, 8, 11 and 15. The differences in scoresfor the rats treated with a vehicle and the rats treated with Compound49 are graphically presented in FIGS. 13-15. The results show thattreatment with Compound 49 normalized the difference in scores betweenthe operated and unoperated paws compared to the continued hyperalgesicvehicle-treated rats. The time taken for the rats to withdraw its paw onthe unoperated (sham) side are graphically presented in FIG. 16. Therats exhibited approximately equal withdrawal latencies on the sham sideregardless of treatment.

Compound 5

The rats were treated with either Compound 5 (50, 30, 10, 3, 1 or 0.3mg/kg) or a vehicle for 12 (or 15) days after sciatic nerve ligation.Thermal pain responses were measured at days 0, 1, 5, 8 and 12 (and 15)The differences in scores for the rats treated with a vehicle and therats treated with Compound 5 are graphically presented in FIGS. 17-22.The results show that treatment with Compound 5 normalized thedifference in scores between the operated and unoperated paws comparedto the continued hyperalgesic vehicle-treated rats. The time taken forthe rats to withdraw its paw on the unoperated (sham) side aregraphically presented in FIG. 23. The rats exhibited approximately equalwithdrawal latencies on the sham side regardless of treatment.

Compound C

The rats received either Compound C (50 mg/kg i.p. daily) or a vehiclestarting 10 days post surgery. Treatment with Compound C dramaticallynormalized the difference scores between the two paws compared to thecontinued hyperalgesic vehicle-treated controls. Normal (unoperated)rats had approximately equal latencies for both paws. This effect wassignificant starting at 11 days of drug treatment and persisted throughto the end of the study (for 21 days of daily dosing). The differencescores are graphically presented in FIG. 8. The results show thatNAALADase inhibition attenuates CCI-associated hyperalgesia.

Example 11 In Vivo Assay of NAALADase Inhibitors on Progression ofNeuropathic Pain in BB/W Models

Compounds D and A

Male BB/W rats (BRI, Mass) spontaneously develop a cell mediatedautoimmune destruction of pancreatic B cells, resulting in onset ofinsulin-dependent (Type I) diabetes (Guberski 1994). These rats havebeen characterized and shown to demonstrate neuropathies withaccompanying neural deficits such as fiber loss and degeneration,changes which are correlative with those seen in peripheral nerve ofhuman diabetic patients (Yagihasi 1997). This renders them valuable forexperimental trials of new compounds for future treatments of this majordisorder. In the present study, Compound D and Compound A were examinedfor their ability to alter the progression of diabetic neuropathy. Therats received daily injection of Compound D or Compound A (10 mg/kgi.p.) or vehicle, starting at the onset of diabetes (hyperglycemia) andup to 6 months thereafter. Another group of non-diabetic rats alsoreceiving vehicle were tested. All animals were continuously monitoredfor body weight, urine volume, blood sugar and glycated haemoglobin. Inthe first month of the study, all animals were tested for thermalnociception in a Hargreaves apparatus, weekly. After the first monththis was done biweekly and then monthly. The testing consists ofdirecting an infrared heat source onto the dorsal surface of the rathindpaw and noting the time taken for the animal to remove its paw (seeHargreaves et al., supra, for a description of the experimental method).Each animal was tested 8 times and the mean withdrawal latency noted.

The results are graphically presented in FIG. 11. The results show thatdiabetic rats displayed a hyperalgesia (shorter response latency)compared to non-diabetic controls. Diabetic drug-treated rats (bothCompound D and Compound A) displayed longer withdrawal latencies thandiabetic vehicle-treated rats, starting after 4 weeks of treatment andpersisting through the six months of treatment.

Nerve conduction velocity was also measured every two weeks through thefirst eight weeks of treatment and every month thereafter through to thesix months of treatment (see De Koning et al., Peptides, Vol. 8, No. 3,pp. 415-22 (1987) for a description of the experimental method). Theresults are graphically presented in FIG. 12. Diabetic animals generallyshowed a reduction in nerve conduction velocity compared to non-diabeticcontrols. However, diabetic animals receiving daily injections ofNAALADase inhibitor (either Compound D or Compound A at a dose of 10mg/kg) showed significantly less severe nerve conduction deficits thandid the diabetic controls receiving vehicle treatment. This was apparentstarting at 8 weeks of treatment and persisted to a similar degreethrough to the six month termination point of the study. Diabeticvehicles, on the other hand, showed a progressive deterioration in nerveconduction velocity from 6 to 16 weeks after start of vehicleadministration which was maintained through to six months.

Example 12 In Vivo Assay of NAALADase Inhibitors on Diabetic Neuropathyin STZ Model

Motor and sensory nerve conduction velocity was also measured inSTZ-diabetic animals after 4, 8 and 12 weeks of treatment (see De Koninget al., supra, for a description of the experimental method). Briefly,stimulating needle electrodes were inserted close to the sciatic andtibial nerves with recording electrodes being placed subcutaneously overthe distal foot muscles, in anesthetized rats. The results aregraphically presented in FIGS. 9A, 9B, 10A and 10B. Diabetic animalsreceiving vehicle showed a significant reduction in both motor andsensory nerve conduction compared to non-diabetic animals. Treatmentwith 10 mg/kg of Compound A daily for 4, 8 and 12 weeks all tended toimprove (increase) both motor and sensory nerve conduction velocities,with a significant improvement being observed after 12 weeks and 8 weeksfor motor and sensory nerve conduction velocity, respectively (FIGS. 9Aand 9B). The lower dose of Compound A tested (1 mg/kg) had similareffects. Treatment of animals with Compound D at either dose alsoincreased both motor and sensory nerve conduction velocities above thatof diabetic controls, significantly so after 12 weeks of treatment forthe 10 mg/kg treatment group (FIGS. 10A and 10B) and at the earlier timeperiods after treatment with the 1 mg/kg dose. Thus, the results showthat NAALADase inhibition alters the progression of diabetic neuropathy.

Example 13 In Vivo Assay of NAALADase Inhibitors on Reversal of DiabeticNeuropathy in STZ Models

General Method for STZ Model—Delayed Dosing

Rats (200 to 225 g) were injected with STZ (70 mg/kg) into the tailvein. Diabetes (>350 mg/dl) was confirmed in all rats, 4 weeks after STZadministration. Rats were left untreated until 35 to 49 days after STZ.Compound D (1, 3, or 10 mg/kg), Compound E (10 mg/kg), or vehicle weredosed daily p.o. following confirmation of hyperalgesia and/or nerveconduction velocity deficits. In separate experiments, onset oftreatment was delayed until 60 to 90 days after STZ administration.Nerve conduction velocity or withdrawal response to thermal stimulationof hind paws was measured at intervals, usually bi-weekly for thermalresponse and monthly for nerve conduction velocity.

General Method for db/db Mice Study

Spontaneously diabetic mice (db/db mice) and non-diabetic littermateswere obtained from Jackson labs. Mice were left untreated until 7 to 8months of age (or after 4 to 5 months of chronic diabetes) and thendosed daily with Compound F (1 mg/kg) p.o. Nerve conduction velocity wasmeasured prior to the onset and after eight weeks of treatment.

Nerve Conduction Velocity Measurements

Sensory and motor nerve conduction velocities were evaluated using themethod of De Koning and Gispen (Peptides 8: 415-422, 1987).Electrophysiological evaluation was carried out within one hour ofdosing. Animals were anesthetized with isoflurane and stimulating needleelectrodes were inserted close to the sciatic nerve at the sciatic notchand the tibial nerve near the ankle. Recording electrodes were placedover the foot muscles. Stimuli were applied and responses recorded.Motor and sensory nerve conduction velocities were calculated bymeasuring the distance between the sciatic notch and ankle sites, andthe latency between the M-wave and the H-reflex.

Thermal Hyperalgesia

Animals were acclimated to the apparatus for at least 5 min. Aninfra-red source was placed under below the plantar surface of the rathind-paw. The intensity of the source was adjusted so that latency fornormal rats was about 10 secs. Animals were tested for thermal responselatency according to the method of Hargreaves et al (Pain 77-88, 1988).Each animal was tested 8 times (4 each. hind limb) and the latency ofresponse recorded automatically to nearest 0.1 sec. An average of thelast 4 measurements for each paw was calculated (8 total measurements)and noted for each rat.

FIG. 31 shows the effect of NAALADase inhibitor (Compound D and CompoundE) treatment on neuropathic pain abnormalities in STZ-diabetic rats. Allrats showed apparent hyperalgesia compared to non-diabetic rats prior toNAALADase inhibitor treatment (5 weeks post STZ). However, within twoweeks of treatment, neuropathic hyperalgesia was reversed towards normalin both NAALADase inhibitor treated groups. This reversal persistedthroughout the subsequent hypoalgesic phase usually seen in prolongeddiabetic-STZ rats, with a reduced hypoalgesic phase displayed inNAALADase treated rats.

FIG. 32 shows the motor nerve conduction velocity measurements in STZdiabetic rats and non-diabetic controls prior to and at time periodsafter NAALADase inhibitor treatment. Within 8 weeks of dosing, bothNAALADase inhibitors Compound D and Compound E reversed the motor nerveconduction velocity towards normal (non-diabetic values). This effectpersisted through 12 weeks of treatment.

FIG. 33 shows sensory nerve conduction velocity deficits, similarlytested. NAALAse inhibitor treatment similarly reversed sensory nerveconduction velocity deficits, significantly so after only 2 weeks oftreatment.

FIG. 34 shows neuropathic pain abnormalities in another experiment wheretreatment with lower doses (1 and 3 mg/kg) of the NAALADase inhibitorCompound D was initiated after 7 weeks of STZ treatment. Significantreduction in pain abnormalities were again apparent with both doses ofCompound D.

FIGS. 35 and 36 show sensory and motor nerve conduction velocity,respectively, in these chronically diabetic STZ rats treated with thelower doses of Compound D. Sensory nerve conduction was significantlyimproved towards normal within 4 weeks of treatment whereas motor nerveconduction remained unimproved by these low doses, even 8 weeks afterdosing.

FIGS. 37 and 38 show sensory and motor nerve conduction velocitymeasurements generated from an external CRO in a similar chronicallydiabetic STZ model, where rats were left untreated until 60 days afterSTZ treatment. Partial reversal of both deficits was again produced byCompound D treatment. FIG. 39 shows the same where treatment was delayedyet further, until 90 days after STZ.

FIG. 40 shows nerve conduction velocity measurements from a geneticmouse model of diabetes, at 6 to 7 months of age (after about 4 monthsof chronic diabetes). A significant impairment in sensory NCV wasapparent at this time. FIG. 41 shows the nerve conduction velocity inthese mice after 8 weeks of treatment with another, more potentNAALADase inhibitor administered at 1 mg/kg daily. Significantimprovement in the sensory nerve conduction was apparent following drugtreatment.

Example 14 Effect of NAALADase Inhibitors on Onset of ALS

The effect of NAALADase inhibitors on the onset of ALS was tested usingthe transgenic mice model of familial amyotrophic lateral sclerosis(FALS), which is detailed in Gurney, M., Annals of Neurology (1996)39:147-157, and otherwise well known in the art. One month oldtransgenic G1H mice were treated with daily intraperitoneal injectionsof a vehicle (50 mM HEPES-buffered saline) or a NAALADase inhibitor (50mg/kg Compound A). Clinical symptoms of the mice were monitored daily.The onset of clinical disease was scored by examining each mouse for itsshaking of limbs when suspended in the air by its tail, cross spread ofspinal reflexes, hindlimb paralysis, body weight and wheel runningactivity.

The results, set forth below in TABLE IV, show that disease onset wasdelayed in mice treated with a NAALADase inhibitor.

TABLE IV EFFECT OF NAALADASE INHIBITOR ON ONSET OF CLINICAL DISEASEDISEASE DISEASE ONSET FOR ONSET FOR COMPOUND A VEHICLE TREATED MICETREATED MICE STUDY (days) (days) DIFFERENCE Study 1 221 189 32 Study 2166 141 25

Example 15 Effect of NAALADase Inhibitor on ALS Survival and ClinicalSymptoms

The effect of NAALADase inhibitors on ALS survival and clinical symptomswas tested using again the transgenic mice model of FALS. One month oldtransgenic G1H mice were treated daily with a vehicle (50 mMHEPES-buffered saline) or a NAALADase inhibitor (30 mg/kg Compound B)p.o. (by oral administration). Clinical symptoms of the mice weremonitored twice a week. Such symptoms included shaking of limbs, gait,dragging of hind limbs, crossing of limbs, righting reflex andmortality. Gait and crossing of limbs were graded on an arbitrary scaleranging from 0 to 3, with 0 representing most normal and 3 representingleast normal, e.g. severest difficulty in walking or crossing limbs.Righting reflex was measured by the time (seconds) it took the mice toright themselves when placed on their sides on a flat surface.

The results, set forth in FIGS. 24-30, show that survival was prolongedand clinical symptoms were attenuated in mice treated with a NAALADaseinhibitor.

Example 16 Protective Effect of NAALADase Inhibitors in Experimental RatGlaucoma

Experimental Protocol

All experiments complied with the Association for Research in Vision andOphthalmology Statement for the Use of Animals in Ophthalmic and VisionResearch. 82 male Brown Norway rats (Rattus norvegicus), each weighingapproximately 250 gm, were treated using procedures approved by theAnimal Care Committee of the Johns Hopkins University School ofMedicine. The rats were housed with a 12 hour light/12 hour dark cycleand fed ad libitum.

Experimental Glaucoma:

Unilateral elevation of intraocular pressure (“IOP”) was produced in 56rats by microinjection of hypertonic saline into episcleral veins,following procedures described in Morrison, J. et al., IOVS (March 1998)39:526-531. Beginning on the day of IOP elevation, the rats were treateddaily with intraperitoneal injections of either a vehicle (23 rats with50 mM HEPES-buffered saline) or a NAALADase inhibitor (11 rats with 10mg/kg of Compound A and 22 rats with 10 mg/kg of Compound B). 11 salinetreated rats, 11 Compound A treated rats and 11 Compound B treated ratswere sacrificed at 8 weeks, and the remaining rats at 12 weeks, afterinitial IOP elevation.

Optic Nerve Transection:

The optic nerve was transected unilaterally in 26 rats underintraperitoneal pentobarbital anesthesia. The conjunctiva was openedwith scissors and the optic nerve exposed by traction on extraocularmuscles. The transection was performed with microscissors 5 mm posteriorto the globe, with specific attention to avoidance of injury to majorocular blood vessels. Immediately after transection, the retina wasexamined ophthalmoscopically to assure that the retinal arterial bloodsupply was not disrupted. The conjunctiva was closed with absorbablesuture and the eye dressed with antibiotic ointment. Beginning on theday of transection, the rats were treated daily with intraperitonealinjections of either a vehicle (9 rats with 50 mM HEPES-buffered saline)or a NAALADase inhibitor (8 rats with 10 mg/kg of Compound A and 9 ratswith 10 mg/kg of Compound B). 5 saline treated rats, 3 Compound Atreated rats and 4 Compound B treated rats were sacrificed at 2 weeks,and the remaining rats at 4 weeks, after transection.

Optic Nerve Counting:

The rats were sacrificed by exsanguination under deep pentobarbitalanesthesia. They were perfused through the heart with 2%paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2,and the eyes with attached optic nerves were removed. A cross-section ofthe optic nerve from both experimental (glaucoma or transection) andcontrol eyes was removed 1.5 mm posterior to the globe, 1 mm inthickness, and post-fixed in 2% osmium tetroxide in buffer. These wereprocessed into epoxy resin, sectioned at 1 micron and stained withtoluidine blue.

The area of the optic nerve cross-section was measured by outlining itsouter border at lox magnification on an image analysis system (UniversalImaging Corp., Westchester, Pa.) with Synsys digital camera andMetamorph software. Three area measurements were taken and the meanvalue was determined. To measure the density and fiber diameterdistributions, images were captured with a 100× phase contrast objectivefrom 10 different areas of each nerve. These were edited to eliminatenon-neural objects and the size of each axon internal to the myelinsheath (its minimum diameter) and the density of axons/square mm werecalculated for each image and nerve. The mean density was multiplied bytotal nerve area to yield fiber number for each nerve. The total fibernumber in glaucoma or transection nerves was compared to the normal,fellow eye of each rat to yield a percent loss value. The number ofaxons counted among the 10 images was an approximately 20% sample of the80-90,000 axons in normal rat nerves. The person measuring axon numberwas masked to the protocol conducted on the nerves.

Results

Experimental Glaucoma:

The mean fiber percent difference in the saline-treated, control ratswas significantly lower in their glaucoma eyes compared to their normaleyes, with a mean fiber loss of 14.44±5.75% (n=11 rats; TABLE V) in the8 week follow-up group, and 8.15±7.84% in the 12 week follow-up group(n=12 rats; TABLE VI).

By contrast, there was no significant loss of fibers in either the 8week or 12 week NAALADase inhibitor-treated rats. The mean percent fiberloss in each NAALADase inhibitor-treated group was statistically lessthan the loss in the saline-treated, control groups (at 8 weeks, p=0.05for Compound A and p=0.02 for Compound B).

TABLE V EXPERIMENTAL GLAUCOMA RESULTS 8 WEEK IOP INTEGRAL PERCENT GROUPN DIFFERENCE FIBER NUMBER DIFFERENCE Compound 11  85 ± 37.5 79156 ±2436* −1.82 ± 2.92   A Compound 11 116 ± 33.2  80785 ± 2121** −0.82 ±2.97   B Control 11 104 ± 26.4 68295 ± 4617  14.44 ± 5.75 

TABLE VI EXPERIMENTAL GLAUCOMA RESULTS 12 WEEK IOP INTEGRAL PERCENTGROUP N DIFFERENCE FIBER NUMBER DIFFERENCE Compound B 11 109 ± 45.290504 ± 1718 −3.21 ± 2.86 Control 12 158 ± 66.5 79827 ± 6783   8.15 ±7.84 IOP Integral Difference = difference in IOP exposure betweenglaucoma eye and normal eye in each rat (mm Hg -- days). PercentDifference = mean percent difference in fiber number between glaucomaand normal eye in each rat (positive value indicates fewer fibers in theglaucoma eye). Differences in IOP Integral Difference are notsignificant (p > 0.05). Differences in Percent Difference betweendrug-treated and saline-treated, control rats at 8 weeks post insult aresignificant (p = 0.05* and p = 0.02**).

Optic Nerve Transection:

The experimental transection data suggest a slowing or rescue ofultimate RGC death in rats treated with NAALADase inhibitors at 2 weeksafter transection. At 2 weeks after transection, both drug-treatedgroups had more remaining RGC axons than did the saline-treated, controlgroup, judged either by absolute number of fibers or percent differencebetween transected eye and normal eye in each rat (TABLE VII). Ratstreated with Compound A and Compound B had, respectively, 3 times andtwice as many remaining axons as the saline-treated rats. All or nearlyall RGC die within the first 2 months after transection, regardless ofany pharmacological treatment. Thus, by 4 weeks after transection, morethan 80% of RGC axons were gone in all groups (TABLE VIII). At 4 weeksafter transection, there were no significant differences between thedrug-treated rats and the saline-treated rats.

TABLE VII OPTIC NERVE TRANSECTION 2 WEEKS SURVIVAL N FIBER NUMBERPERCENT DIFFERENCE Compound A 3 26,426 ± 23,025 65.3 ± 30.9 Compound B 419,550 ± 11,383 75.3 ± 14.8 Control 5 8,220 ± 9,337 90.2 ± 10.7

TABLE VIII OPTIC NERVE TRANSECTION 4 WEEKS SURVIVAL N FIBER NUMBERPERCENT DIFFERENCE Compound A 5 13,599 ± 7,868 82.4 ± 8.9 Compound B 5 5,162 ± 5,017 93.4 ± 6.2 Control 4 10,449 ± 8,157  86.9 ± 10.6 PercentDifference = mean percent difference in fiber number between glaucomaand normal eye in each rat (positive value indicates fewer fibers in theglaucoma eye). Differences in Percent Difference between drug-treatedand saline-treated, control rats are not statistically significant (p =0.05).

Example 17 Neuroprotective Effect of NAALADase Inhibitors in TransgenicMouse Model of Huntington's Disease

Behavioral Testing (Rotarod)

Transgenic HD mice of the N171-82Q strain and non-transgenic littermateswere treated with NAALADase inhibitor Compound B (30 mg/kg) or a vehiclefrom 10 weeks of age. The mice were placed on a rotating rod(“rotarod”). The length of time at which the mouse fell off the rotarodwas recorded as a measure of motor coordination. FIG. 42 shows thattransgenic HD mice treated with Compound B stayed longer on the rotarodthan similar transgenic HD mice treated with a vehicle. The treatmentwith Compound B had no effect on the rotarod performance of normalnon-HD mice.

The total distance traveled by the mice was also recorded as a measureof overall locomotion. FIG. 43 shows that while the vehicle treated HDmice demonstrated the lowest mean locomotor score, the treatment withNAALADase inhibitor had no apparent effect on overall locomotion.

Survival

The effects of Compound B and vehicle on the survival of transgenic HDmice (N171-82Q) were evaluated. Thirteen mice (six male and sevenfemale) were assigned to the Compound B treatment group, and fourteenmice (six male and eight female) were assigned to the vehicle treatmentgroup. Treatment was continued until all the mice died.

FIG. 44 shows the survival distributions over time by treatment group.The median survival time is 184 days for the Compound B treatment group,and 158.5 days for the vehicle treatment group. Although the Compound Btreatment group had a longer median survival time than the vehicletreatment group, the difference is not statistically significant(p-value=0.07).

FIGS. 45 and 46 show the survival distributions over time by treatmentgroup and sex. When analyzing the results specific to sex, female micetreated with Compound B had significantly prolonged survival time(p-value=0.03) compared to their vehicle treated counterparts. Withinthe vehicle treatment group, the males have better survival times thanthe females although this trend was not observed in the Compound Btreatment group. The data suggest that sex may influence survivaldistributions over time.

Example 18

A patient is suffering from any disease, disorder or condition whereNAALADase levels are altered, including any of the diseases, disordersor conditions described above. The patient may then be administered aneffective amount of a compound of the invention. It is expected thatafter such treatment, the patient would not suffer any significantinjury due to, would be protected from further injury due to, or wouldrecover from the disease, disorder or condition.

All publications, patents and patent applications identified above areherein incorporated by reference, as though set forth herein in full.

The invention being thus described, it will be apparent to those skilledin the art that the same may be varied in many ways without departingfrom the spirit and scope of the invention. Such variations are includedwithin the scope of the following claims.

We claim:
 1. A compound of formula III

or a pharmaceutically acceptable equivalent of said compound, wherein: Xand Y are independently —CR⁵R⁶—, —O—, —S— or —NR—, provided that atleast one of X and Y is/are —CR⁵R⁶—; A¹, A², A³, A⁴ and A⁵ areindependently hydrogen, C₁-C₉ alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl,heteroaryl, carbocycle, heterocycle, C₁-C₉ alkoxy, C₂-C₉ alkenyloxy,phenoxy, benzyloxy, hydroxy, halo, cyano, isocyano, —COOR⁷, —COR⁷,—NR⁷R⁸, —SR⁷, —SOR⁷, —SO₂R⁷, —SO₂(OR⁷), —(C═O)NR⁷R⁸,—(C═O)NR⁷(CH₂)_(n)COOH, —NR⁷(C═O)R⁸ or —(CH₂)_(n)COOH, or any adjacenttwo of A¹, A², A³, A⁴ and A⁵ form with the benzene ring a fused ringthat is saturated or unsaturated, aromatic or non-aromatic, andcarbocyclic or heterocyclic, said heterocyclic ring containing 1 or 2oxygen, nitrogen and/or sulfur heteroatom(s); n is 1-3; R, R¹, R², R³,R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, C₁-C₉ alkyl, C₂-C₉alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle or heterocycle; andsaid alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle,alkoxy, alkenyloxy, phenoxy, benzyloxy, and fused ring are independentlyunsubstituted or substituted with one or more substituent(s); providedthat if A¹, A² and A³ are each hydrogen, and A⁴ and A⁵ are each —COOH,then A⁴ is ortho to A⁵ and provided that if Y is —CR⁵R⁶—, then at leastone of A¹, A², A³, A⁴ and A⁵ is/are independently phenoxy, benzyloxy,aryl, heteroaryl, carbocycle or heterocycle that is substituted with oneor more substituent(s). and provided that at least one of A¹, A², A³, A⁴and A⁵ are not hydrogen.
 2. The compound of claim 1, wherein: Y is —O—,—X— or —NR—; A¹, A², A³, A⁴ and A⁵ are independently hydrogen, C₁-C₄alkyl, C₁-C₂ alkoxy, hydroxy, halo, —COOH, —COR⁷, —NR⁷(C═O)R⁸ or—(CH₂)COOH; and R⁷ and R⁸ are independently hydrogen or methyl.
 3. Thecompound of claim 1, wherein: Y is —CR⁵R⁶—; A¹, A², A³ and A⁴ are eachhydrogen; and A⁵ is phenoxy, benzyloxy, aryl, heteroaryl, carbocycle orheterocycle, wherein said phenoxy and benzyloxy are substituted with—COOH, and said aryl, heteroaryl, carbocycle and heterocycle aresubstituted with one or more substituent(s) selected from the groupconsisting of cyano and —COOH.
 4. The compound of claim 1, wherein saidcompound is selected from the group consisting of:4-(2-cyanophenyl)-alpha-(3-mercaptopropyl)-benzenepropanoic acid;3-(1-carboxy-4-mercaptobutoxy)-benzoic acid;5-mercapto-2-phenoxy-pentanoic acid;2-(3,5-dimethoxyphenoxy)-5-mercapto-pentanoic acid;2-(3-hydroxyphenoxy)-5-mercapto-pentanoic acid;3-(1-carboxy-4-mercaptobutoxy)-benzeneacetic acid;4-(1-carboxy-4-mercaptobutoxy)-benzeneacetic acid;2-(3-acetylphenoxy)-5-mercapto-pentanoic acid;2-[3-(acetylamino)phenoxy)-5-mercapto-pentanoic acid;2-(4-acetylphenoxy)-5-mercaptopentanoic acid;3-(1-carboxy-4-mercaptobutoxy)-4-methoxy-benzoic acid;2-(1-carboxy-4-mercaptobutoxy)-benzoic acid;4-(1-carboxy-4-mercaptobutoxy)-benzoic acid;3-(1-carboxy-4-mercaptobutoxy)-4-chloro-benzoic acid;3-(1-carboxy-4-mercaptobutoxy)-4-fluoro-benzoic acid;5-mercapto-2-(phenylthio)-pentanoic acid;3-[1-carboxy-4-mercaptobutyl)thio]-benzoic acid;3′-(2-carboxy-5-mercaptopentyl)-[1,1′-biphenyl]-3-carboxylic acid;3′-(2-carboxy-5-mercaptopentyl)-[1,1′-biphenyl]-2-carboxylic acid;3-(2-carboxyphenoxy)-alpha-(3-mercaptopropyl)-benzenepropanoic acid;4-(2-carboxyphenoxy)-alpha-(3-mercaptopropyl)-benzenepropanoic acid;4′-(2-carboxy-5-mercaptopropyl)-[1,1′-biphenyl]-2-carboxylic acid;3-(1-carboxy-4-mercaptobutoxy)-5-(1,1-dimethylethyl)-benzoic acid;3-[(1-carboxy-4-mercaptobutyl)thio]-5-(1,1-dimethylethyl)-benzoic acid;3-[(1-carboxy-4-mercaptobutyl)amino]-5-(1,1-dimethylethyl)-benzoic acid;and pharmaceutically acceptable equivalents.
 5. The compound of claim 1,wherein said compound is an enantiomer or an enantiomer-enrichedmixture.
 6. A method for inhibiting NAALADase enzyme activity, treatinga glutamate abnormality, effecting a neuronal activity, treating aprostate disease, treating cancer, inhibiting angiogenesis or effectinga TGF-β activity, comprising administering to a mammal in need of suchinhibition, treatment or effect, an effective amount of a compound ofclaim
 1. 7. A method for detecting a disease, disorder or conditionwhere NAALADase levels are altered, comprising: (i) contacting a sampleof bodily tissue or fluid with an effective amount of a compound ofclaim 1, wherein said compound binds to any NAALADase in said sample;and (ii) measuring the amount of any NAALADase bound to said sample,wherein the amount of NAALADase is diagnostic for said disease, disorderor condition.
 8. A method for detecting a disease, disorder or conditionwhere NAALADase levels are altered in a mammal, comprising: (i) labelinga compound of claim 1 with an effective amount of an imaging reagent;(ii) administering to said mammal an effective amount of the labeledcompound; (iii) allowing said labeled compound to localize and bind toNAALADase present in said mammal; and (iv) measuring the amount ofNAALADase bound to said labeled compound, wherein the amount ofNAALADase is diagnostic for said disease, disorder or condition.
 9. Adiagnostic kit for detecting a disease, disorder or condition whereNAALADase levels are altered, comprising a compound of claim 1 labeledwith a marker.
 10. A pharmaceutical composition comprising: (i) aneffective amount of a compound of claim 1; and (ii) a pharmaceuticallyacceptable carrier.